Studies on dextranases. IV. Mode of action of dextranase D1 on oligosaccharides

The products of action of a purified, extracellular endo-dextranase D₁, isolated from a new species of Pseudomonas, on pure isomaltose oligosaccharides have been investigated. Reduced and tritiated oligosaccharides have also been studied, and a model is postulated for the enzyme active-site, based on substrate specificity.     

Studies on dextrans and dextranases. XI. The structure of a dextran elaborated by Leuconostoc mesenteroides NRRL B-1299

The synthesis of di-(6-deoxy-β-D-allofuranose) 1,5′:1′,5-dianhydride (8) from 6-deoxy-D-allose is described. Periodate oxidation of 8, followed by borohydride reduction and acetylation, yielded a crystalline 2,4,7,9-tetra(acetoxymethyl)-5.10-dimethyl-1,3,6,8-tetraoxecane (3).     

Modes of action of intracellular dextranase and three oligoglucanases from Pseudomonas UQM733

The action patterns have been studied of a purified, intracellular dextranase and three intracellular alpha-D-glucosidases from Pseudomonas UQM733 on pure isomalto-oligosaccharides. The glucosidases have optimal activity on isomaltotetraose and are therefore classified as oligoglucanases. They have been used to determine the structure of two branched isomalto-oligosaccharides obtained by enzymic degradation of dextran.     

Histones H2a, H2b, H3, and H4 form a tetrameric complex in solutions of high salt.

In 2 M NaCl, histones H2b, H2a, H3, and H4 form a heterotypic tetrameric complex made up of one chain of each histone. This complex has been analyzed by hydrodynamic techniques. It is indistinguishable from histones in chromatin by its resistance to trypsin, pattern of reactivity with 125I. and ability to form specific crosslinked products after treatment with formaldehyde. It is proposed that this complex is responsible for protecting the small DNA fragments produced by exhausting nuclease digestion of nuclei and that on the average two of these complexes protect the larger 180-200 base pair unit produced by partial treatment of nuclei with nuclease.     

Structures of the oligosaccharides from the enzymic hydrolysis of hemicellulose by a hemicellulase of Ceratocystis paradoxa.

And endo-hemicellulase (HC-II) of Ceratocystis paradoxa degraded spear-grass hemicellulose B to a series of mixed oligosaccharides. Four neutral oligosaccharides (AraXyl2, AraXyl3, Xyl2, and Xyl3), isolated by preparative paper chromatography, were shown by enzymic and methylation techniques to constitute a series of beta-(1 leats to 4)-D-xylose and O-alpha-L-arabinofuranosyl-(1 leads to 3)-O-beta-D-xylopyranosyl-(1 leads to 4)-O-beta-D-xylopyranosyl-(1 leads to 4)-D-xylose, respectively, the latter being a new compound.     

Energy-liked reactions in photosynthetic bacteria. X. Solubilization of the membrane-bound energy-linked inorganic pyrophosphatase of Rhodospirillum rubrum.

The energy-linked membrane-bound inorganic pyrophosphatase of $\text{Rhodospirollum}$$\text{rubrum}$, G-9, has been solubilized with good yield from chromatophores using cholate in the presence of MgCl₂. The enzyme has been partially purified using ammonium sulfate fractionation and gel chromatography. After fractionation the enzyme requires phospholipid for activity. The solubilized enzyme is specific for PP_i and requires Mg²⁺ for activity as has been found for other PP_iases.     

3′and 4′ and equatorial amino and hydroxy derivatives of neamine

All eight of the possible amino and hydroxyl derivatives at the 3′ and 4′ positions of neamine were prepared. The compounds are either 3′-deoxy or 4′-deoxy analogs having axial or equatorial amino or hydroxyl groups at the other position. The position and configuration of the hydroxyl groups from the reduction of the two 3′, 4′-epoxides, 11 and 12, were established. The 3′-keto and 4′-keto derivatives (26 and 23) and also the corresponding oximino acetates, 33b and 35b, were reduced and the structures of the products determined. Hydrolytic cleavage of the exo-methylene epoxides at the 3′ and 4′ positions gave the diols, 47 and 45. N.m.r. data for the 3′ and 4′ acetates and acetamides are presented.     

A crystallographic determination of a chemical structure: 6-amino-10-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine, an example of an unusual D-ribose conformation.

The crystal and molecular structure of 6-amino-10-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine has been determined by single crystal X-ray diffraction methods. The crystals are triclinic, of noncentric space group Pl, with cell dimensions a equals 5.434 (5), b equals 12.269 (19), c equals 4.574 (4) A, alpha equals 92.3 (1), beta equals 94.0 (1), gamma equals 95.3 degrees (1) and Z equals 1. The structure has been refined to an R value of 0.049 (Rw equals 0.063), by use of counter measured intensity data for 1063 observed reflections. The pyrimidopyrimidine ring is planar. The sugar moiety is in the envelope conformation with O-1'-endo (0E), and there is an intramolecular hydrogen bond (2.58 A) (O-3'-H-O3'...O-2'). All oxygen atoms except O-1' ring oxygen-atom are involved in hydrogen bonding. The pyrimidopyrimidine rings lie in planes 3.4 A apart.     

Digestion of polysaccharide constituents of tropical pasture herbage in the bovine rumen. IV. The hydrolysis of hemicelluloses from spear grass by cell-free enzyme systems from rumen fluid

Hemicelluloses have been isolated from spear grass (Heteropogon contortus), before and after digestion in the rumen, and separated into linear and branched fractions. Similar fractions have also been obtained from a pasture sample and from the faeces fibre of a steer fed on the same pasture. The rates of hydrolysis of all of these hemicellulose fractions have been determined in the presence of extracellular enzymes from rumen fluid and of enzymes liberated by disruption of rumen microorganisms. The oligosaccharide products of such enzymic degradations have been partially identified. The results confirm that the incomplete digestion of hemicelluloses in the rumen is due to physical protection (e.g. by lignin), rather than to structural differences between different components of the hemicelluloses. There is no difference between rates of digestion of branched and linear hemicelluloses, and previous results which indicated such differences were probably caused by presence of a readily digested glucan in linear hemicellulose fractions.     

The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

A uniparentally inherited mutation affecting photophosphorylation in Chlamydomonas reinhardi.

A photosynthetically incompetent mutant strain of $\text{Chlamydomonas}\text{reinhardi}$ shows a uniparental mode of inheritance, a substantial level of photosynthetic electron transport activities, in whole cells and in a cell free system, but a negligible level of photosynthetic phosphorylation activity in a cell free system.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

Studies on bacterial cell wall inhibitors: II. Inhibition of peptidoglycan synthesis in vivo and in vitro by amphomycin

Amphomycin has been reported by the present authors to be a selective inhibitor of cell wall peptidoglycan synthesis in Bacillus cereus T (ōmura, S., Tanaka, H., Shinohara, M., ōiwa, R. and Hata, T. (1975) Chemotherapy 5, 365–369). Investigations were carried out to clarify the target of amphomycin.Amphomycin (10 μg/ml) lysed growing cells of B. cereus T, and inhibited peptidoglycan synthesis, accompanied by accumulation of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc) peptides. The nucleotide precursors that accumulated in cells of Staphylococcus aureus FDA 209P in the presence of amphomycin were identified as UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala, UDP-MurNAc-L-Ala and UDP-MurNAc. In the experiments using a particulate enzyme system of Bacillus megaterium KM, amphomycin inhibited the polymerization of UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) and UDP-N-acetylglucosamine, and also inhibited the formation of lipid intermediates, but did not inhibit the cross-linking, the last step of peptidoglycan synthesis. Unlike bacitracin, amphomycin did not lyse protoplasts of B. megaterium KM.We conclude that the site of action of amphomycin is the formation of MurNAc-(pentapeptide)-P-P-lipid from MurNAc-pentapeptide and undecaprenol (lipid) phosphate.     

Synthesis of 3-O Substituted D-Mannoses

1,2,4,6-Tetra-O-acetyl-3-O-benzyl-α-D-mannopyranose (7) was obtained in good yield from 3,4,6-tri-O-benzyl-1,2-O-(1-methoxyethylidene)-β-D-mannopyranose (1) by acetolysis. Hydrogenolysis of 7 afforded 1,2,4,6-tetra-O-acetyl-α-D-mannopyranose which is a versatile intermediate for the preparation of other 3-O-substituted D-mannoses, such as 3-O-methyl-D-mannose and 3-O-α-D-mannopyranosyl-D-mannose. 3,4-Di-O-methyl-D-mannose was readily prepared from 1,2,6-tri-O-acetyl-3,4-di-O-benzyl-α-D-mannopyranose, which was also obtained from 1 by controlled acetolysis.