Evidence that the cytosolic activity of 3-hydroxybutyrate dehydrogenase in chicken liver isL-3-hydroxyacid dehydrogenase

Classical fractionation studies showed that chicken liver contains two enzymes which can oxidize DL-3-hydroxybutyrate. The cytosolic enzyme is specific for the L-(+) isomer and accounts for 60% of the total activity. The mitochondrial activity is specific for the D-(?) isomer and accounts for 40% of the total activity. Kinetic studies showed that L-gulonic acid is a competitive inhibitor of the enzyme. We conclude that the cytosolic enzyme is the previously described L-3-hydroxyacid dehydrogenase.     

Selenium derivatives of L-arabinose, D-ribose,and D-xylose

Attempted cyclization of 2,3,4-tri-O-methyl-5-seleno-L-arabinose dimethyl acetal in acidic solution gave the corresponding diselenide. Intramolecular attack by the selenobenzyl group at C-5 of 5-O-p-tolylsulfonyl-L-arabinose dibenzyl diseleno-acetal resulted in the formation of benzyl 1,5-diseleno-L-arabinopyranoside. Similarly, 2,3,5-tri-O-methyl-4-O-p-tolylsulfonyl-D-xylose dibenzyl diselenoacetal gave benzyl 2,3,5-tri-O-methyl-1,4-diseleno-L-arabinofuranoside, and 2,3,4-tri-O-acetyl-5-O-p-tolylsulfonyl-D-xylose (or ribose) dibenzyl diselenoacetal gave benzyl 2,3,4-tri-O-acetyl-1,5-diseleno-D-xylo- (or ribo-)pyranoside. The glycosylic benzylseleno group was removed from the pyranoside with mercuric acetate, but attempted deacetylation of the product led to decomposition and not to the expected 5-seleno-D-xylopyranose.     

Proton magnetic resonance spectra of D-gluco-oligosaccharides and D-glucans

The p.m.r. spectra of some D-gluco-oligosaccharides and D-glucans in deuterium oxide were studied with respect to the anomeric proton. In (1→2)-linked glucobioses, the effect of change in configuration of the hydroxyl group at C-1 on the chemical shifts of the glycosidic proton is noted. Equilibrium mixtures of (1→2)-linked glucobioses contained more α-anomer than did the other examples, despite the cis configuration of substituents at C-1 and C-2. Some D-glucans were investigated with regard to the degree of branching, although solubility was a limitation.     

Further characterization of L-β-hydroxyacid dehydrogenase from Drosophila

L-β-Hydroxyacid dehydrogenase (L-β-hydroxyacid--NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-β-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-β-hydroxyacid dehydrogenase IgG revealed a single L-β-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

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.     

The orientation of d-β-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane

d-β-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and ‘inside-out’ submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A₂ digestion than from mitochondria. Conclusive evidence that d-β-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome $\text{c}$. The arrangement of d-β-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that d-β-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.     

Growth of Rhodopseudomonas capsulata on l- and d-malic acid

d-malate replaced l-malate in supporting both photosynthetic (anaerobic, light) and heterotrophic (aerobic, dark) growth of Rhodopseudomonas capsulata. Growth rates and cell yields were nearly equivalent with both enantiomorphs. Addition of glucose to malate culture media increased the growth rate and doubled the cell yield of heterotrophic cultures, but had little effect on photosynthetic cultures. Aerobically-grown cells showed a higher level of substrate-dependent oxygen uptake with l-malate than with d-malate. This preference for l-malate occured even in cells grown on d-malate. No malic racemase activity was detected in extracts of heterotrophically- or photosynthetically-grown cells.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

Phenylalanine ammonia-lyase: Mirror-image packing of d- and l-phenylalanine and d- and l-transition state analogs into the active site

The binding of substrate and product analogs to phenylalanine ammonia-lyase (EC 4.3.1.5) from maize has been studied by a protection method. The ligand dissociation constants, K_L, were estimated from the variation with [L] of the pseudo-first-order rate constants for enzyme inactivation by nitromethane. The phenylalanine analogs d- and l-2-aminooxy-3-phenylpropionic acid showed K_L, values over 20,000-fold lower than the K_m for l-phenylalanine. From these and other K_L values it is deduced that when the enzyme binds l-phenylalanine the structural free energy stored in the protein is higher than when it binds the superinhibitors. Models for binding d- and l-phenylalanine and the superinhibitors are described. The enantiomeric pairs are considered to have similar K_L values because they pack into the active site in a mirror-image relationship. If the elimination reaction approximates to the least-motion course deduced on stereoelectronic grounds, the mirror-image packing of the superinhibitors into the active site mimics the conformation inferred for a transition state in the elimination. It appears, therefore, that structural changes take place in the enzyme as the transition state conformation is approached causing stored free energy to be released. This lowers the activation free energy for the elimination reaction and accounts for the strong binding by the above analogs.     

Detection and properties of l-histidinol dehydrogenase in wheat germ

The presence and partial characterization of the properties of l-histidinol dehydrogenase (EC 1.1.1.23), the enzyme catalysing the last step in the pathway of histidine biosynthesis, has been described in higher plants for the first time. The activity has been found in cell-free extracts from wheat germ, turnip root, radish root and squash fruit. The enzyme has been partially purified and characterized from extracts of acetone powders of wheat germ. DEAE-cellulose chromatography revealed two peaks of histidinol dehydrogenase activity. In one there was a rapid reduction of NAD⁺ in the absence of histidinol; however, the rate was stimulated by the addition of histidinol. The rate in the absence of substrate became quite low after several min and the histidinol-dependent rate was then easily observed. The second peak of activity did not reduce NAD⁺ unless l-histidinol was present in the assay mixture. The K^ms for l-histidinol and NAD⁺ were determined for this latter enzyme. The values obtained at saturating concentrations of the other substrate were l-histidinol, 8.8 μM and NAD⁺, 0.14 mM. The product of the dehydrogenase reaction was histidine as determined by paper chromatography.     

Preparation of some acylated D-arabino-hexopyranosuloses and 4-deoxy-D-glycero-hex-3-enopyranosuloses

Oxidation of 1,3,4,6-tetra-O-benzoyl-α- and β-D-glucopyranose gave the tetra-O-benzoyl-α- and -β-D-arabino-hexopyranosuloses (3α and β), from which benzoic acid was readily eliminated to give the anomeric tri-O-benzoyl-4-deoxy-D-glycero-hex-3-enopyranosuloses (4α and β). The anomeric 1-O-acetyl-tri-O-benzoyl-D-arabino-hexopyranosuloses (7α and β) were obtained as very unstable syrups which readily lost benzoic acid. Treatment of tetra-O-benzoyl-2-O-benzyl-D-glucopyranose (1) with hydrogen bromide gave 3,4,6-tri-O-benzoyl-α-D-glucopyranosyl bromide (5) in one step.     

D-Galactose 6-phosphate: anomeric distribution and analysis of its synthesis from D-galactose and polyphosphoric acid

D-Galactose 6-phosphate as synthesized by direct phosphorylation of D-galactose with polyphosphoric acid is contaminated with two of its positional isomers. These were separated from D-galactose 6-phosphate and from each other, and identified as D-galactose 3- and 5-phosphate by enzymic, chromatographic, and mass-spectral analysis. The previous misidentification of these isomers as furanose forms of D-galactose 6-phosphate has led to erroneous reports concerning the anomeric distribution of D-galactose 6-phosphate. The anomeric distribution of D-galactose 6-phosphate in a purified preparation was determined by gas-liquid chromatography and ¹³C-n.m.r. spectroscopy to be 32% α-pyranose, 64% β-pyranose, and no more than 4% furanose anomers.     

The oxidation of terminal D-galactofuranose residues of a galactan and a glycoprotein by a D-galactose Oxidase preparation from Dactylium dendroides

A galactan, isolated from the unicellular organism Prototheca zopfii, and a glycoprotein from a hyphal cell-wall fraction of the fungus Pithomyces chartarum have been oxidised by a D-galactose oxidase preparation from Dactylium dendroides. The oxidised polymers were subsequently reduced with sodium borotritide. The site of oxidation was identified as C-6 of non-reducing D-galactofuranosyl residues in both polymers.     

α-D-mannosidase and α-D-galactosidase from protein bodies of Lupinus angustifolius cotyledons

Two forms of p-nitrophenyl α-D-mannosidase and p-nitrophenyl α-D-galactosidase were purified from the protein bodies of mature Lupinus angustifolius seeds. A MW of 300 000 was calculated for both α-mannosidase A and B with K_m = 1.92 and 2.70 mM and activation energies of 10.9 and 10.8 kcal/mol, respectively. α-Galactosidase I and II had MWs of 70800 and 17000 with K_m = 0.282 and 0.556 mM and activation energies 17.7 and 11.5 kcal/mol, respectively. The enzymes had acid pH optima and were inhibited by various metal ions, carbohydrates and glycoproteins. They were able to release free sugar from several putative natural substrate oligosaccharides and the Lupinus storage glycoprotein, α-conglutin.     

Twinning in crystals of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase

The coenzyme-bound form of human skeletal muscle d-glyceraldehyde-3-phosphate dehydrogenase has been shown to crystallize in the space group C2 and not C222₁ as previously reported. The unit cell contains two tetrameric molecules with the dimer of molecular weight 72,000 as the crystallographic asymmetric unit. The recorded X-ray intensity distribution clearly indicates the presence of non-crystallographic 2-fold axes perpendicular to the crystallographic 2-fold axis showing that the subunits are arranged with near perfect 222 symmetry.Isomorphous derivatives of the enzyme have been prepared and the heavy atom positions defined in complete agreement with the C2 space group assignment. Further confirmation that the space group is C2 and not C222₁ comes from the 3.5 Å resolution electron density map of the human enzyme, which appears almost identical to that of the lobster holo-enzyme where no such space group ambiguity exists.     

Synthesis of D-ristosamine and its derivatives

A convenient preparative route involving eleven steps starting from D-glucose is described for the synthesis of D-ristosamine (15) hydrochloride. Methyl 2-deoxy-β-D-arabino-hexopyranoside, prepared from 3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-arabino-hex- 1-enitol, was benzylidenated, and the product mesylated to give methyl 4,6-O-benzylidene-2-deoxy-3-O-methylsulfonyl-β-D-arabino-hexopyranoside. Azidolysis of this compound and subsequent opening of the 1,3-dioxane ring with N-bromosuccinimide gave methyl 3-azido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-βD-ribo-hexopyranoside. Simultaneous reduction of the azido and bromo groups gave a mixture that was benzoylated to give methyl N,O-dibenzoyl-β-D-ristosaminide and then hydrolyzed to 15 hydrochloride (3-amino-2,3,6-trideoxy-D-ribo-hexopyranose hydrochloride).     

Glycans from streptococcal cell-walls: the molecular structure of an antigenic diheteroglycan of D-glucose and L-rhamnose from Streptococcus bovis

The monosaccharide sequence and glycosidic bond-types have been determined for an antigenic diheteroglycan of D-glucose and L-rhamnose from the cell wall of Streptococcus bovis, strain C3, by use of an integrated analytical scheme based on methylation analysis, periodate oxidation, oxidation with chromium trioxide, enzymic hydrolysis, and chemical degradation. A typical molecule of the glycan consists of a main chain of L-rhamnosyl residues and isomaltose side-chains, with 16 repetitions of the structure, -α-L-rhamnosyl-(1→3)-[α-D)-glucosyl-(1→6)-α-D-glucosyl-(1→2)]-α-L-rhamnosyl-(1→2)-α-L-rhamnosyl-, linked alternately by α-L-(1→3) and α-L-(1→2) linkages. The isomaltose side-chains of the glycan are the immunodeterminant groups. The new antigenic glycan is ideally suited for use in the preparation of anti-isomaltose antibodies, which should be of value in the detection of other antigens having isomaltose determinants.