Enzymatic β-galactosidation of β-xylopyranosides

Summary The disaccharides formed by enzymatic transfer of the -D-galactopyranosyl residue fromo-nitrophenyl -d-galactopyranoside to -d-xylopyranosides have been identified. The influence of different factors on the yields of the disaccharides obtained was evaluated. Significant changes in selectivity were observed when -galactosidase fromE. coli was used instead of -galactosidase fromA. oryzae.     

Possible Identity of β-Xylosidase and β-Glucosidase of Chaetomium trilaterale

The nature of the active site of Chaetomium trilaterale β-xylosidase catalyzing the hydrolysis of β-d-glucopyranoside and β-d-xylopyranoside was investigated by kinetic methods. On experiments with mixed substrates, such as phenyl β-d-xylopyranoside and phenyl β-d-glucopyranoside, the kinetic features agreed very closely with those features theoretically predicted for a single active site of the same enzyme catalyzing the hydrolysis of these two kinds of substrates.

Both the β-glucosidase and β-xylosidase activities were strongly inhibited by glucono-1,5-lactone and nojirimycin (5-amino-5-deoxy-d-glucopyranose). β-Xylosidase activity was inhibited non-competitively by the two inhibitors, but β-glucosidase activity was competitive. Methyl β-d-xylopyranoside, methyl β-d-glucopyranoside, 1-thiophenyl β-d-xylopyranoside, and 1-thiophenyl β-d-glucopyranoside poorly inhibited both activities. Methyl β-d-xylopyranoside inhibited the β-xylosidase activity competitively but the β-glucosidase activity was non-competitive, whereas methyl β-d-glucopyranoside inhibited the β-xylosidase activity non-competitively but the β-glucosidase activity was competitive. 1-Thiophenyl β-d-xylopyranoside and 1-thiophenyl β-d-glucopyranoside behaved as competitive inhibitors.

From these results, it was concluded that the β-xylosidase and β-glucosidase activities reside in one catalytic site, and this suggests that there might be two kinetically distinct binding sites in the active center of the same enzyme.     

Prediction of β-Turns

Kohonen's self-organization model, a neural network model, is applied to predict the -turns in proteins. There are 455 -turn tetrapeptides and 3807 non--turn tetrapeptides in the training database. The rates of correct prediction for the 110 -turn tetrapeptides and 30,229 non--turn tetrapeptides in the testing database are 81.8% and 90.7%, respectively. The high quality of prediction of neural network model implies that the residue-coupled effect along a polypeptide chain is important for the formation of reversal turns, such as -turns, during the process of protein folding.     

Folding of β-Hairpins

Identification of the factors governing the formation of -structure independently of the rest of the protein is important for understanding the folding process of protein into a unique native structure. It has been shown that some -hairpins can fold autonomously into native-like structures, either in aqueous solution or in the presence of an organic co-solvent. Our aim is to review recent theoretical and experimental studies of folding of -structures.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Synthesis of β-Tubanol

Synthesis of β-tubanol (VIa) was achieved by the bromination of dihydro-β-tubanol acetate (VIIb) followed by dehydrobromination and the subsequent hydrolysis. 2,2-Dimethyl-5-hydroxy-chromanone as well as its derivatives (IV) and the corresponding chromanols (V) and chromans (VII) were also prepared.     

Prediction of β-Turns

Kohonen's self-organization model, a neural network model, is applied to predict the β-turns in proteins. There are 455 β-turn tetrapeptides and 3807 non-β-turn tetrapeptides in the training database. The rates of correct prediction for the 110 β-turn tetrapeptides and 30,229 non-β-turn tetrapeptides in the testing database are 81.8% and 90.7%, respectively. The high quality of prediction of neural network model implies that the residue-coupled effect along a polypeptide chain is important for the formation of reversal turns, such as β-turns, during the process of protein folding.     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases

p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H₂ ¹⁸O to liberate galactose containing ¹⁸O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.     

Biotransformation of β-ketosulfides to produce chiral β-hydroxysulfoxides

The biotransformations of a series of substituted phenylthio-2-propanone and benzylthio-2-propanone were carried out using Helminthosporium sp. NRRL 4671, Mortierella isabellina ATCC 42613, or Rhodococcus erythropolis IGTS8. Several products gave microbial oxidation of sulfide to sulfoxide and reduction of carbonyl to secondary alcohol, producing β-hydroxysulfoxides in medium to high enantiomeric and diastereomeric purities. Fungal biotransformations using Helminthosporium sp. and M. isabellina resulted in the opposite sulfoxide configurations of various β-hydroxysulfoxide products.     

Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.     

Syntheses of Several β-l-Rhamnopyranosides

To investigate the substrate specificity of β-l-rhamnosidase, the following β-l-rhamnopyranosides were synthesized: 1-(β-l-rhamnopyranosyl)-dl-glycerol (1), methyl β-l-rhamnopyranoside (2), methyl 2-O-(β-l-rhamnopyranosyl)-β-d-glucopyranoside (3) and methyl 2-O-β(β-l-rhamnopyranosyl)-α-l-arabinopyranoside (4). The synthesis of 3 was performed using l-quinovose with neighboring group participation, which lead stereoselectively to the β-l-quinovoside. The 2-OH of the l-quinovo-unit was selectively deblocked, oxidized to the keto group, and then stereoselectively reduced, whereby 3 was produced.     

Nonopioid effect of β-endorphin

This review presents the generalized literature data and the results of our own research of the nonopioid effect of β-endorphin, an opioid neuropeptide interacting not only with opioid but also with nonopioid (insensitive to the opioid antagonist naloxone) receptors. The roles of the hormone and its receptors in regulation of the immune, nervous, and endocrine systems are discussed. The effect of neuromediator on the immune system mediated by both opioid and nonopioid receptors is considered in detail. The data on distribution and function of the nonopioid β-endorphin receptor in human and animal organisms are presented. All available data on the characteristics of the nonopioid β-endorphin receptor obtained by means of radioligand analysis are given. The discussed information is supposed to extend our conceptions of the role of β-endorphin in mammals and to be of extensive use in medicine and pharmacology.     

Synthesis of derivatives of methyl β-sophoroside

Selective tritylation of methyl β-sophoroside (1) and subsequent acetylation gave the 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-trityl derivative, which was O-detritylated, and the product p-toluenesulfonylated, to give methyl 3,4,2′,3′,4′-penta-O-acetyl-6,6′-di-O-p-tolylsulfonyl-β-sophoroside (4) in 63% net yield. Compound 4 was also obtained in 69% yield by p-toluenesulfonylation of 1, followed by acetylation. Several, 6,6′-disubstituted derivatives of 1 were synthesized by displacement reactions of 4 with various nucleophiles. Treatment of 4 with sodium methoxide afforded methyl 3,6:3′,6′-dianhydro-β-sophoroside. Several 6- and 6′-monosubstituted derivatives of 1 were prepared, starting from the 4,6-O-benzylidene derivative of 1.     

Preparation of Aryl β-d-Mannopyranoside Derivatives via β-d-arabino-Hexopyranosiduloses

Sequential oxidation and reduction of aryl 4, 6-O-benzylidene-β-d-glucosides with dimethyl sulfoxide-phosphorus pentoxide mixture (DMSO–P₂O₅) and sodium borohydride were carried out as a new means for the preparation of aryl β-d-mannopyranoside derivatives. p-Nitrophenyl 4, 6-O-benzylidene-β-d-mannopyranoside was obtained in 22% yield from the corresponding glucoside 3-O-acetate, whereas from the unprotected acetal, 4, 6-O-benzylidene acetals of the corresponding mannoside and alloside were isolated in the yields of 6.7 and 2.1%, respectively. Similarly, phenyl 4, 6-O-benzylidene β-d-mannoside, alloside, and altroside were obtained from the corresponding glucoside in 2.2, 0.8 and 2.1% yields, respectively.     

Separation of β-d-galactosidases in rabbit tissues: Genetics of neutral β-d-galactosidase

Three different types of β-d-galactosidase (EC 3.2.1.23) could be distinguished in rabbit tissues using electrophoretic procedures. (1) Acid β-d-galactosidase with a low mobility and maximal activity atpH 3–5 was found in the particulate fraction of various tissue homogenates. This enzyme hydrolyzed 4-methylumbelliferyl-d-galactoside, but no activity against other glycoside substrates could be demonstrated. The enzyme was inhibited by galactono-(1 → 4)-lactone. (2) Lactose-hydrolyzing β-d-galactosidase with an intermediate mobility was found only in juvenile small intestine. Most of the activity was found in the particulate fraction of the cell. The enzyme hydrolyzed several other synthetic glycoside substrates besides lactose. It was most active atpH 5–6 and strongly inhibited by glucono-(1 → 5)-lactone but not much affected by galactono-(1 → 4)-lactone. (3) Neutral β-d-galactosidase with a fast mobility and maximal activity atpH 6–8 was found in the soluble fraction of homogenates from liver, kidney, and small intestine. This enzyme also showed a broad substrate specificity; it possessed activity against aryl-β-d-glucoside, -fucoside, and -galactoside substrates but not against lactose. The enzyme was strongly inhibited by glucono-(1 → 5)-lactone and (less) by galactone-(1 → 4)-lactone. Neutral β-d-galactosidase and neutral β-d-glucosidase (EC 3.2.1.21) are probably identical enzymes in the rabbit. Individual variation, in both electrophoretic mobility and activity, was found for neutral β-d-galactosidase. Genetic analysis of the electrophoretic variants revealed that two alleles at an autosomal locus are responsible for this variation. This investigation was supported in part by Public Health Service Grant RR-00251 from the Division of Research Resources and by funds of the University of Utrecht.