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.     

Approaches to the synthesis of sugar thiolactones

Evidence is presented that aldonolactones undergo “alkyl-oxygen” fission when attacked by thionucleophiles. The reaction of 2,3-O-isopropylidene-d-ery-throno-1,4-lactone with potassium thioacetate gives 2,3-O-isopropylidene-4-thio-d-erythrono-1,4-lactone, the first example of a thiolactone of an aldonic acid. Deacetylation of 5-S-acetyl-2,3-O-isopropylidene-5-thio-d-ribono-1,4-lactone is accompanied by partial migration of sulphur from C-5 to C-4; a mechanism involving an intermediate 5,6-episulphide is suggested.     

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.     

Purification,properties, and mode of action of hemicellulase II produced by Ceratocystis paradoxa

An extra-cellular endo-hemicellulase (HC-II) from a culture isolate of the fungal plant pathogen Ceratocystis paradoxa (CP₂) was purified 147-fold by ammonium sulphate precipitation, DEAE-Sephadex chromatography, iso-electric focusing at pH 3–10, and gel-permeation chromatography. The resulting enzyme preparation, which contained traces of invertase, gave a single protein-band on disc electrophoresis at pH 8.4, and was active towards sucrose, hemicellulose, and carboxymethylcellulose (CMC). HC-II randomly degraded hemicelluloses from several different sources, to xylose and to arabinose-xylose and xylose oligosaccharides of d.p. 3–6 and 2–5, respectively, and also produced a degraded hemicellulose which precipitated from the digest solution. The precipitated hemicellulose contained less arabinose and uronic acid than the original hemicellulose. When redissolved by alkali-treatment, it was susceptible to further degradation by hemicellulases HC-I and HC-II. CMC was degraded by HC-II, mainly to D-glucose and cellobiose, with trace amounts of unidentified higher oligosaccharides, while cellobiose remained unattacked. Xylotriose (Xyl₃) was the lowest homologue of the xylose oligosaccharides attacked by HC-II at a significant rate, yielding xylobiose [Xyl₂; β-D-Xylp-(1→4)-D-Xyl] and xylose. AraXyl₃AraXyl₅ were mainly hydrolysed to AraXyl₂, xylose, and Xyl₂ or Xyl₃. HC-II had a temperature optimum of 80°, and was stable for 1 h at temperatures up to 70°. The pH optimum was 5.1, and HC-II was stable between pH 5–10. The K_m was 0.267 mg of hemicellulose B/ml. The effects of mercury(II) ions and high concentrations of xylose on the activity of HC-II were also investigated.     

Temporary site in the anti-tryptic fragment from adzuki-bean proteinase inhibitor II

The anti-tryptic fragment, derived from adzuki-bean proteinase inhibitor II, was subjected to limited proteolysis by trypsin at pH 2.9 for 48 h. Three peptide bonds, Lys-Ser, Arg-Cys and Arg-Asp, were split, inactivating the fragment. The temporary site, the point of inactivation against trypsin, was concluded to be Arg-Cys, since the Lys-Ser bond is the reactive site and the tripeptide (Asp)_3′ released by the cleavage of the Arg-Asp bond, should not affect the inhibitory activity. This effective bond, corresponding to Arg³²-Cys³³ of inhibitor II, was possibly more exposed to the enviromental solvent by cuting down the anti-chymotryptic domain from the parent inhibitor.     

Studies on dextranases. 3. Insolubilization of a bacterial dextranase

Ammonium hydroxide treatment of 1,6:2,3-dianhydro-4-O-benzyl-β-D-mannopyranose, followed by acetylation, gave 2-acetamido-3-O-acetyl-1,6-anhydro-4-O-benzyl-2-deoxy-β-D-glucopyranose which was catalytically reduced to give 2-acetamido-3-O-acetyl-1,6-anhydro-2-deoxy-β-D-glucopyranose (6), the starting material for the synthesis of (1→4)-linked disaccharides bearing a 2-acetamido-2-deoxy-D-glucopyranose reducing residue. Selective benzylation of 2-acetamido-1,6-anhydro-2-deoxy-β-D-glucopyranose gave a mixture of the 3,4-di-O-benzyl derivative and the two mono-O-benzyl derivatives, the 4-O-benzyl being preponderant. The latter derivative was acetylated, to give a compound identical with that just described. For the purpose of comparison, 2-acetamido-4-O-acetyl-1,6-anhydro-2-deoxy-β-D-glucopyranose has been prepared by selective acetylation of 2-acetamido-1,6-anhydro-2-deoxy-β-D-glucopyranose.Condensation between 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide and 6 gave, after acetolysis of the anhydro ring, the peracetylated derivative (17) of 2-acetamido-2-deoxy-4-O-β-D-glucopyranosyl-α-D-glucopyranose. A condensation of 6 with 3,4,6-tri-O-acetyl-2-deoxy-2-diphenoxyphosphorylamino-α-D-glucopyranosyl bromide likewise gave, after catalytic hydrogenation, acetylation, and acetolysis, the peracylated derivative (21) of di-N-acetylchitobiose.     

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.     

Purification and characterisation of two endo-(1→3)-β-d-glucanases from Telescopium telescopium

The crystalline style of the gastropod Telescopium telescopium contains two (1→3)-β-d-glucanases and a β-d-glucosidase. The two glucanases (I and II) have been purified and shown to be endo-enzymes. Both enzymes attack laminarin, carboxymethylpachyman, and lichenin, but have no action towards carboxymethyl-cellulose. The main products of hydrolysis of laminarin are d-glucose and β-(1→3)-linked oligosaccharides of d.p. 2, 3, and 4. Glucanases I and II are similar to each other, although they differ in molecular weight and kinetic properties.     

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.     

An affinity column for renin

An affinity column for renin was prepared making use of the strong affinity of pepstatin for renin. Pepstatin is an N-acylated pentapeptide from $\text{Actinomycetes}$ with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy-6-methylheptanoic acid. This peptide was coupled to aminoethylated polyacrylamide gel either directly with the water-soluble carbodiimide, 1-ethyl-3-(dimethylaminopropyl) carbodiimide, or through the N-hydroxy succinimide ester. Submaxillary renin was selectively retained by a small column of the gel and was eluted by a salt gradient to produce a highly pure material. This column was also effective for the purification of renal renin.     

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.