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.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

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.     

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.     

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.     

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.     

Peroxisomal β-ketothiolase

β-Ketothiolase was found in rat liver peroxisomes that were isolated by sucrose density gradient centrifugation. The presence of dithiothreitol was essential for measuring the peroxisomal thiolase, but dithiothreitol had little effect upon the thiolase activity in the mitochondria or cytosol. Dithiothreitol was not used during the isolation procedures. Acetoacetyl CoA and β-ketolauryl CoA, which was synthesized chemically, were used as substrates. The peroxisomal thiolase was active with β-ketolauryl CoA but had almost no activity with acetoacetyl CoA as the substrate. Thiolase activities in the mitochondrial and cytosolic fractions utilized both long- and short-chain substrates. The thiolases in the various fractions bound to and were purified by chromatography on calcium phosphate gel cellulose columns. The peroxisomal thiolase did not bind to phosphocellulose, whereas the mitochondrial and cytosolic activities could be chromatographed on phosphocellulose. Peroxisomal β-ketolauryl CoA thiolase activity was inhibited about 20% by 25 mm Mg²⁺, and more activity was measured in a phosphate than in a Tris buffer. In control rat livers, the total activity of β-ketolauryl CoA thiolase was 3.4 μmol/min per gram of liver in the peroxisomes and 4.9 μmol/min per gram of liver in the mitochondria, which indicates that peroxisomes contain the capacity for 40% of the total thiolase activity associated with β-oxidation systems. In addition, 6.3 μmol/min per gram of liver of β-ketolauryl CoA thiolase was found in the soluble fraction and chromatographed differently from the peroxisomal enzyme on phosphocellulose. Clofibrate in the rat diet for 6 or 21 days resulted in about a 15-fold increase in peroxisomal thiolase when assayed with β-ketolauryl CoA and somewhat less of an increase in the other fractions.     

Novel mutations involving βI-, βIIA-, or βIVB-tubulin isotypes with functional resemblance to βIII-tubulin in breast cancer

Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the β subunit of tubulin, which is a heterodimer of α and β subunits. β-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the βIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of β-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their β-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the βI-, βIIA-, or βIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the βIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of βI- or βIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, βI, βIIA, and βIVB have up to seven mutations to the corresponding residues in βIII-tubulin. Given that βIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate β-tubulin isotypes to make them acquire some of the pro-survival properties of βIII-tubulin. These “hybrid” tubulins, combining the sequences and/or properties of two wild-type tubulins (βIII and either βI, βIIA, or βIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.     

GRB10 regulates β-cell mass by inhibiting β-cell proliferation and stimulating β-cell dedifferentiation

Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1 cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling.     

Splitting of naphthol AS-BI β-galactopyranoside by acid β-galactosidase

Summary -Galactosidase hydrolyses naphthol AS-BI -galactopyranoside in a variety of rat and mouse organs using freeze-dried cryostate sections, hexazonium-p-rosaniline for simultaneous coupling and long time incubation. In comparison with the indolyl and naphthyl derivate the splitting rate of the naphthol AS compound is far lower.     

Characterization of β-lapachone and methylated β-cyclodextrin solid-state systems

The purpose of this research was to explore the utility of β cyclodextrin (βCD) and β cyclodextrin derivatives (hydroxypropyl-β-cyclodextrin [HPβCD], sulfobutylether-β-CD [SB\CD], and a randomly methylated-β-CD [RMβCD]) to form inclusion complexes with the antitumoral drug, β-lapachone (βLAP), in order to overcome the problem of its poor water solubility. RMβCD presented the highest efficiency for βLAP solubilization and was selected to develop solid-state binary systems. Differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), Fourier transform infrared (FTIR) and optical and scanning electron microscopy results suggest the formation of inclusion complexes by both freeze-drying and kneading techniques with a dramatic improvement in drug dissolution efficiency at 20-minute dissolution efficiency (DE_20-minute 67.15% and 88.22%, respectively) against the drug (DE_20-minute 27.11%) or the βCD/drug physical mixture (DE_20-minute 27.22%). However, the kneading method gives a highly crystalline material that together with the adequate drug dissolution profile make it the best procedure in obtaining inclusion complexes of RMβCD/βLAP convenient for different applications of βLAP. Published: July 27, 2007     

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.     

Lp-Typensystem und β-Lipoprotein-Konzentration

Serum -lipoprotein content after Heiskell et al. and Lp(a) type were determined and compared for 258 healthy unrelated adults. The mean -lipoprotein content was found to be significantly greater (255,2 mg-%) in Lp(a+) sera than in Lp(a-) sera (217,9 mg-%).     

Cysteine S-conjugate β-lyases

Summary. Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate-containing enzymes that catalyze β-elimination reactions with cysteine S-conjugates that possess an electron-withdrawing group attached at the sulfur. The end products of the β-lyase reaction are pyruvate, ammonium and a sulfur-containing fragment. If the sulfur-containing fragment is reactive, the parent cysteine S-conjugate may be toxic, particularly to kidney mitochondria. Halogenated alkenes are examples of electrophiles that are bioactivated (toxified) by conversion to cysteine S-conjugates. These conjugates are converted by cysteine S-conjugate β-lyases to thioacylating fragments. Several cysteine S-conjugates found in allium foods (garlic and onion) are β-lyase substrates. This finding may account in part for the chemopreventive activity of allium products. This review (1) identifies enzymes that catalyze cysteine S-conjugate β-lyase reactions, (2) suggests that toxicant channeling may contribute to halogenated cysteine S-conjugate-induced toxicity to mitochondria, and (3) proposes mechanisms that may contribute to the antiproliferative effects of sulfur-containing fragments eliminated from allium-derived cysteine S-conjugates.     

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography

A new screening method for β-(1,3–1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.     

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.     

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.