Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

Regioselective reaction of thiols with catechol estrogens and estrogen-O-quinones

Incubations of [3H]estradiol and [3H]2-hydroxyestradiol (2-OHE2) with rat liver microsomes and mushroom tyrosinase were carried out in the presence of glutathione and 2-mercaptoethanol. A ratio of about 3.5:1 for the C-4 and C-1 thioether conjugates of 2-OHE2 was observed. Chemical reaction of estradiol-2, 3-O-quinone with various thiols showed that alkyl and phenyl thiols gave about a 1:1 ratio of C-4 to C-1 thioethers. However, reaction of the O-quinone with 4-nitrothiophenol gave a C-4/C-1 ratio of 0.25 while 4-bromothiophenol gave a C-4/C-1 ratio of 4.0. These studies suggest that the regioselectivity of the reaction of thiols with estrogen catechols and O-quinones may be dependent on the nature of the thiol compounds and less on steric hindrance.     

Synthesis and monoamine oxidase B substrate properties of 1-methyl-4-heteroaryl-1,2,3,6-tetrahydropyridines

Six analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP, (1)] bearing various heteroaryl groups at C-4 were synthesized and examined for their monoamine oxidase B substrate properties. The C-4 substituents include the 1-ethylpyrrol-2-yl, 1-propylpyrrol-2-yl, 1-isopropylpyrrol-2-yl, 1-cyclopropylpyrrol-2-yl, 3-ethylfuran-2-yl and 3-ethylthien-2-yl groups. The results provide information concerning steric and polar interactions between the C-4 substituent and the active site of MAO-B that are transmitted to the position of oxidation at C-6 of the tetrahydropyridinyl moiety.     

Secondary structure of charge isomers of myelin basic protein before and after phosphorylation

Human myelin basic protein (MBP) was fractionated into several of its charge isomers (components). Of these, the secondary structures of four isomers before and after phosphorylation have been studied by circular dichroism (CD). None of the four showed any alpha-helical structure. All of the components showed varying amounts of beta-structure, random structure, and turns. Component 1 (C-1), the most cationic of the components, showed 13%; component 2 (C-2) had 19%; C-3, 17%; and C-4, 24% of beta-structure. Each of the four components was phosphorylated with protein kinase C, from human brain. The extent of phosphorylation varied considerably from 2.8 +/- 0.6 mol of PO4/mol of protein in C-1 to 5.2 +/- 0.8 mol of PO4/mol of protein in C-4. The effect of phosphorylation on the secondary structure was to induce beta-structure in all the components. The largest change in beta-structure was in C-1 and the least in C-4. The surprising result is that although the components were phosphorylated to different extents, the amount of beta-structure in all four components increased to a final proportion of 35-40%. Treatment of phosphorylated C-1 with acid phosphatase removed 50% of the total radioactivity. Although the remainder represented approximately 1 mol of PO4/mol of protein, the proportion of beta-structure was unaltered. We concluded that a single phosphorylation site identified as residues 5-13 represented a critical size for stabilization of beta-structure of MBP in solution and that phosphorylation at the other sites had little influence on secondary structure.     

Purification and characterization of cytosolic aldolase from carrot storage root.

The time courses of incorporation of 13C from 13C-labelled glucose or acetate into cerebral amino acids (glutamate, glutamine and 4-aminobutyrate) and lactate were monitored by using 13C-n.m.r. spectroscopy. When [1-13C]glucose was used as precursor the C-2 of 4-aminobutyrate was more highly labelled than the analogous C-4 of glutamate, whereas no label was observed in glutamine. A similar pattern was observed with [2-13C]glucose: the C-1 of 4-aminobutyrate was more highly labelled than the analogous C-5 of glutamate. Again, no labelling of glutamine was detected. In contrast, [2-13C]acetate labelled the C-4 of glutamine and the C-2 of 4-aminobutyrate more highly than the C-4 of glutamate; [1-13C]acetate also labelled the C-1 and C-5 positions of glutamine more than the analogous positions of glutamate. These results are consistent with earlier patterns reported from the use of 14C-labelled precursors that led to the concept of compartmentation of neuronal and glial metabolism and now provide the possibility of distinguishing differential effects of metabolic perturbations on the two pools simultaneously. An unexpected observation was that citrate is more highly labelled from acetate than from glucose.     

Multiple molecular forms of human plasma butyrylcholinesterase. I. Apparent molecular parameters and broad pattern of the quaternary structure (author's transl)]

Apparent molecular parameters (molecular weights, sedimentation constants, partial specific volumes, free electrophoretic mobilities and isoelectric points) of the four molecular forms C-1, C-2, C-3 and C-4 of human plasma butyrylcholinesterase (EC 3.1.1.8) have been demonstrated by polyacrylamide gel electrophoresis methods and centrifugation in sucrose gradient. The C-1 component is the monomeric form of the enzyme )Mr = 84 800 +/- 5800). All the forms are partially interconvertible and C-1, C-3, C-4 are size isomers corresponding to the monomer, dimer and tetramer of the enzyme. An estimation of the general shape of these forms attempted from electrophoretic and hydrodynamic parameters suggests that they are prolate ellipsoids. The C-4 component in which the axial ratio is at least equal to 8 appears to be arranged as a dimer of dimers (C-3)2 in which the two units are associated in a quasi-linear fashion. The C-2 component is composed of C-1 associated with an inactive smaller subunit, which is responsible for its specific electrical properties (mobility and isoelectric point).     

The C4-dicarboxylic acid pathway of photosynthesis. Identification of intermediates and products and quantitative evidence for the route of carbon flow

1. When leaves with the C(4)-dicarboxylic acid pathway of photosynthesis are exposed to (14)CO(2) the major labelled compounds formed, in order of labelling, are dicarboxylic acids, 3-phosphoglycerate, bexose phosphates and sucrose. During the present studies several quantitatively minor intermediates were identified and their labelling behaviour is described. 2. The pattern of labelling of dihydroxyacetone phosphate, fructose 1,6-diphosphate and ribulose di- and mono-phosphates during radiotracer pulse-chase experiments was consistent with their operation as intermediates in the pathway of carbon dioxide fixation. 3. Serine, glycine, alanine and glutamate had labelling patterns typical of products secondary to the main flow of carbon. 4. The mechanism of the transfer of label from C-4 of dicarboxylic acids to C-1 of 3-phosphoglycerate was also examined. Evidence consistent with pyruvate being derived from C-1, C-2 and C-3 of oxaloacetate, and for a relationship between ribulose 1,5-diphosphate and the acceptor for the C-4 carboxyl group, was obtained. 5. Evidence is provided that, under steady-state conditions, essentially all the label incorporated from (14)CO(2) into C-1 of 3 phosphoglycerate enters via C-4 of the dicarboxylic acids. These and other studies indicated that the route via dicarboxylic acids is essentially the sole route for entry of carbon into 3-phosphoglycerate.     

Synthesis and characterisation of dextran and pullulan sulphate

Dextrans and pullulans of different molar masses in the range of 10(4)-10(5) g/mol were sulphated via a SO3-pyridine complex. The degree of substitution achieved was DS = 2.4 and DS = 1.4 for dextran sulphate and DS = 2.0 and DS = 1.4 for pullulan sulphate, respectively. Confirmation of sulphation was given by FTIR spectroscopy. Asymmetrical S=O and symmetrical C-O-S stretching vibrations were detected at 1260 and 820 cm(-1). Reactivity of the polysaccharide C-atoms was determined by 13C NMR spectroscopy: For dextran this was C-3 > C-2 > C-4, while for pullulan it was C-6 > C-3 > C-2 > C-4.     

Mode of cleavage of the c-c bonds in methyl pento- and hexo-pyranosides with periodate

The course of oxidation reactions of three methyl pentopyranosides and five methyl hexopyranosides with periodate was studied by simultaneous determination of the conjugated aldehydes in the products. It was found that the C-3-C-4 bonds in all of these glycosides were cleaved rapidly, but the velocity of subsequent cleavage of the C-2-C-3 bonds depended on the configuration of the substituent groups on the pyranose ring. Equatorial C-1 methoxyl and C-2 hydroxyl groups favored cleavage of the C-2-C-3 bonds more than did the corresponding axial groups, whereas the equatorial C-5 hydroxymethyl group suppressed C-2-C-3-bond-cleavage. The 4-isomeric glycosides were oxidized at the same rate, without regard to the configuration at C-4.     

Assignment of 13C Chemical Shifts of α-D-Ribonucleoside Sugar Carbons by 1J(CH) Coupling Constants

Abstract

The ¹J(CH) coupling constant of C-1 in nucleosides is increased compared to those of the other carbons of the sugar moiety. Applying this to several D-ribonucleosides the signals C-4′/C-1′of these a-anomers are reversed to those of the 8-counterparts (C-1′/C-4′). This phenomenon and the broadening of the C-3′ signal compared to that of C-2′ establishes the seauence C-4′,1′,2′,3′,5′ (increasing field) for a number of α-D-ribonucleosides.     

Microbial transformations of flavanone and 6-hydroxyflavanone by Aspergillus niger strains

Flavanone (1) and 6-hydroxyflavanone (2) were subjected to transformation by means of Aspergillus niger strains (one wild and three UV mutants). For both substrates the biotransformation resulted in reduction of the carbonyl group (products 5 and 7) and dehydrogenation at C-2 and C-3 (3 and 8). Additionally, for flavanone (1) reduction of C-4 together with hydroxylation at C-7 (6) and dehydrogenation at C-2, C-3 along with hydroxylation at C-3 (4) were observed.     

Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones

Sixteen new and one known metabolites 4-20 were obtained by incubation of tibolone (1) and hydroxytibolones (2 and 3) with various fungi. Their structures were elucidated by means of a homo and heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by 2D NOESY experiment. Metabolites of tibolone (1) exhibited significant inhibitory activities against α-glucosidase and tyrosinase enzymes. Hydroxylations at C-6, C-10, C-11, C-15 positions and α,β-unsaturation at C-1/C-2, C-4/C-5 showed potent inhibitory activities against these enzymes.     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside

The cellulose model compound methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (6) was synthesised in high overall yield from methyl beta-D-cellobioside. The compound was crystallised from methanol to give colourless prisms, and the crystal structure was determined. The monoclinic space group is P2(1) with Z=2 and unit cell parameters a=6.6060 (13), b=14.074 (3), c=9.3180 (19) A, beta=108.95(3) degrees. The structure was solved by direct methods and refined to R=0.0286 for 2528 reflections. Both glucopyranoses occur in the 4C(1) chair conformation with endocyclic bond angles in the range of standard values. The relative orientation of both units described by the interglycosidic torsional angles [phi (O-5' [bond] C-1' [bond] O-4 [bond] C-4) -89.1 degrees, Phi (C-1' [bond] O-4 [bond] C-4 [bond] C-5) -152.0 degrees] is responsible for the very flat shape of the molecule and is similar to those found in other cellodextrins. Different rotamers at the exocyclic hydroxymethyl group for both units are present. The hydroxymethyl group of the terminal glucose moiety displays a gauche-trans orientation, whereas the side chain of the reducing unit occurs in a gauche-gauche conformation. The solid state (13)C NMR spectrum of compound 6 exhibits all 14 carbon resonances. By using different cross polarisation times, the resonances of the two methyl groups and C-6 carbons can easily be distinguished. Distinct differences of the C-1 and C-4 chemical shifts in the solid and liquid states are found.     

角蛋白降解菌的筛选及其UV诱变选育

从长年堆积废羽毛的土壤,屠鸡场下水道、池塘底污泥中分离出一株角蛋白降解菌C-1,摇瓶发酵测得其角蛋白酶活达35.6U,经形态观察和生理生化鉴定该菌株属于芽孢杆菌属.对菌株进行Uv诱变,在照射时间为100 s下得到一株产角蛋白酶活较高突变株C-1-4,其酶活比原始出发菌株提高了122%,且遗传性状稳定.     

A novel stereospecific synthesis of 14C labeled 1-glutamic acid

A stereospecific synthesis of 4-14C-1-glutamic acid was completed in five steps from sodium 2-14C-acetate. The morpholine derived enamine of ethyl pyruvate was reacted with ethyl 2-14C-bromoacetate to give after hydrolysis diethyl 4-14C-2-oxoglutarate. The 2-oxoglutarate was reacted with hydroxylamine hydrochloride to give diethyl 4-14C-2-hydroxyiminoglutarate which was then reduced with a LiAlH4, (-)-N-methylephedrine and 3,5-dimethylphenol mixture to give 4-14C-1-glutamic acid. The 4-14C-1-glutamic acid was used in investigations into the biosynthesis of gamma-lactones in sherries.     

Effect of natural and synthetic benzyl benzoates on calmodulin

The present investigation describes the effect of the spasmolytic benzylbenzoates 1-9 from Brickellia veronicifolia on CaM using a functional in vitro enzymatic assay. Bovine brain PDE1 was used as a monitoring enzyme. The most active natural inhibitors of the system CaM-PDE1 were benzyl benzoates 3-5, which inhibited the activity of PDE1 in a concentration-dependent manner. In addition, three series of analogs of compound 4, compounds 10a-32a, were prepared and assayed. The benzyl benzoates from the first series, namely 10a-24a, possess no substituents on ring B but different number and position of hydroxyl or methoxy groups in ring A. The second group (25-32a), on the other hand, possesses an A ring identical to that on compound 4, but different substituents in Ring B. The most active compounds were 14a, 15a and 30a. These compounds were two to six times more potent than chlorpromazine, a well known CaM inhibitor. Benzyl benzoates 14a and 15a have methoxyl groups at C-2/C-4 and C-3/C-4 in ring A, respectively; while 30a, in addition to the methoxyl groups at C-2/C-6 of ring A, hold a benzoyloxy moiety at C-3' of ring B. Kinetic studies revealed that compounds 3, 4, 14a, 15a and 30a behave as competitive CaM antagonists.     

Phosphorylation and sulfation of oligosaccharide substrates critically influence the activity of human beta1,4-galactosyltransferase 7 (GalT-I) and beta1,3-glucuronosyltransferase I (GlcAT-I) involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans

We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human beta1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galbeta1-3Gal (Gal2-Gal1) on the activity and specificity of beta1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Galbeta1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Galbeta1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp243 and Lys317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.     

Proton and carbon-13 nuclear magnetic resonance spectroscopy of diastereoisomeric 3- and 17 beta-tetrahydropyranyl ether derivatives of estrone and estradiol

Protection of 3- and 17 beta-hydroxyl groups of estrone and estradiol as tetrahydropyranyl ether derivatives led to mixtures of 2'(R)- and 2'(S)-diastereoisomers which were separated by crystallization (3-tetrahydropyranyl ethers), or by thin-layer chromatography (17-tetrahydropyranyl ethers), and characterized by 1H and 13C nuclear magnetic resonance (NMR). Assignments for NMR signals of estradiol 3,17 beta-ditetrahydropyranyl ether were facilitated by comparison with those of its 15 zeta, 16 zeta-dideuterio analog and by 2D 1H-13C heteroshift correlation experiments. Diastereoisomers of 3-tetrahydropyranyl ether derivatives could be identified through the 13C NMR doublet signals of the anomeric C-2' and the aromatic C-4 carbon atoms in CDCl3. Diastereoisomers of 17-tetrahydropyranyl ether derivatives were recognized from characteristic modifications of 1H NMR signals of H-2', H-6', H-1, H-17, and 18-CH3 protons as well as from the 13C NMR doublet signals corresponding to C-2', C-4', C-6', C-12, C-13, C-16, and C-17 carbon atoms. Low-temperature experiments showed a splitting of the C-2', C-6', and C-17 13C NMR signals of each of the two 17-tetrahydropyranyl ether isomers. The downfield signal (equatorial conformer) of the three resulting doublets was more intense for the 17-tetrahydropyranyl ether 2'(S)-isomer, whereas the upfield signal (axial conformer) was more intense for the 2'(R)-isomer.     

Preparation and some properties of rat skeletal-muscle polyribosomes

1. A series of d-galactose derivatives substituted at C-1 and C-6 were tested for active accumulation by everted segments of hamster and rat intestine. 2. d-Galactose and 6-deoxy-6-fluoro-d-galactose were accumulated far more rapidly than 6-deoxy- and 6-chloro-6-deoxy-d-galactose, and this is interpreted as due to hydrogen-bonding at C-6 during the transport process. 3. 6-Bromo-6-deoxy- and 6-deoxy-6-iodo-d-galactose were not actively transported, indicating that the allowed size of substituent at C-6 lies between that of chlorine and bromine atoms. 4. Similar results were obtained at C-1. Both methyl alpha-d-galactopyranoside and methyl beta-d-galactopyranoside were well transported, but methyl beta-d-thiogalactopyranoside and 1-deoxy-d-galactose were not transported; d-galactopyranosyl fluoride was transported, but only poorly. Again hydrogen-bonding is suggested. 5. It is proposed that d-glucose is the ideal structure for active transport and that binding occurs at C-1, C-2, C-3, C-4 and C-6. Loss of two or more of these bonds usually causes loss of active transport. 6. By plotting Lineweaver-Burk plots of the rates of transport of the galactose derivatives, the apparent V and K(m) values were obtained. With hamster intestine both these values were very reproducible. Contrary to expectation, V varied for different sugars. 7. The K(i) of some of the analogues modified at C-1 and C-6 was determined with methyl alpha-d-glucoside as substrate. 8. An attempt to alkylate the carrier by using methyl 3,4-anhydro-alpha-d-galactoside was unsuccessful. There was no evidence that this compound was bound to the carrier.