Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

α- and β-Amylases in seed germination

During the first 24 h of germination of wheat seeds, starch is hydrolysed by free β-amylase. In the next 24 h, some amount of inactive form of β-amylase is converted into active form and this together with α-amylase synthesizedde novo brings about the hydrolysis of starch. The amount of α-amylase is greater in seeds with embryo intact than with embryo excised after 24 h hydration. However, at later stages of seed germination α-amylase becomes predominant and the activity of β-amylase steadily diminishes.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling

The functional selectivity of adrenergic ligands for activation of β1- and β2-AR (adrenoceptor) subtypes has been extensively studied in cAMP signalling. Much less is known about ligand selectivity for arrestin-mediated signalling pathways. In the present study we used resonance energy transfer methods to compare the ability of β1- and β2-ARs to form a complex with the G-protein β-subunit or β-arrestin-2 in response to a variety of agonists with various degrees of efficacy. The profiles of β1-/β2-AR selectivity of the ligands for the two receptor-transducer interactions were sharply different. For G-protein coupling, the majority of ligands were more effective in activating the β2-AR, whereas for arrestin coupling the relationship was reversed. These data indicate that the β1-AR interacts more efficiently than β2-AR with arrestin, but less efficiently than β2-AR with G-protein. A group of ligands exhibited β1-AR-selective efficacy in driving the coupling to arrestin. Dobutamine, a member of this group, had 70% of the adrenaline (epinephrine) effect on arrestin via β1-AR, but acted as a competitive antagonist of adrenaline via β2-AR. Thus the structure of such ligands appears to induce an arrestin-interacting form of the receptor only when bound to the β1-AR subtype.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Enzymatic synthesis of cyclohexyl-α and β-d-glucosides catalyzed by α- and β-glucosidase in a biphase system

Two secondary alcohol glucosides, cyclohexyl-α-d-glucoside and cyclohexyl-β-d-glucoside, were synthesized via the condensation reaction of cyclohexanol with d-glucose in a biphase system catalyzed by α-glucosidase and β-glucosidase, respectively. The effects of pH, water content, glucose concentration and metal ions on the yield of glucosides were studied. The optimum catalytic conditions established for α-glucosidase was 25% (v/v) water content, 2.5 mol/L glucose concentration and pH 2.0, and for β-glucosidase was 30% (v/v) water content, 2.0 mol/L glucose and pH 5.0. The maximum yield of glucoside was 13.3 mg/mL for cyclohexyl-α-d-glucoside and 8.9 mg/mL for cyclohexyl-β-d-glucoside. Synthesis progress was monitored by TLC and quantitatively analyzed by pre-derived capillary gas chromatography (GC). The retention time was 12.34 min for the α isomer and 12.96 min for the β isomer, respectively. With an anomeric purity of more than 99.5%, the two glucosides display excellent site-specific catalysis by α- and β-glucosidase. Herein, we present a general method to produce anomerically pure glucosides via a one-step bio-reaction in a biphase system. This method could potentially be applied in glucosylation of primary and secondary alcohols or other reactions requiring glucosylation.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

Photorespiration and β-carboxylation in brown macroalgae

Photorespiration has been assayed in a variety of marine macroalgae by the Warburg oxygen inhibitory effect and the percentage¹⁴C-labeling of glycine and serine. It was found that particularly the members of the brown macroalgae examined, such asFucus andLaminaria (Phaeophyceae), show a rather weak response to high O₂ partial pressure. This observation is attributed to the appreciably high potential for -carboxylation via PEP-carboxykinase, generally occurring in brown seaweeds.     

Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Ethylene induced chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and -1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [³⁵S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the -1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native -1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and -1,3-glucanase. A putative -1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of -1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for -1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and -1,3-glucanase are regulated co-ordinately at the level of mRNA.Abbreviations poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Lusitropic effects of α- and β-adrenergic stimulation in amphibian heart

The effects of and -adrenergic stimulation in amphibian superfused hearts and ventricular strips were studied. Superfusion with 3×10^–8 M isoproterenol produced a positive inotropic effect, as detected by a 92±24% increase in the maximal rate of contraction and a positive lusitropic effect characterized by a decrease in both the ratio (23±5%) and the half relaxation time (t_1/2) (19±4%). The mechanical behavior induced by the -agonist was associated with an increase in the intracellular cAMP levels from control values of 173±19 to 329±28 nmol/mg wet tissue. Hearts superfused with³²P in the presence of isoproterenol showed a significant increase in Tn 1 phosphorylation (from 151±13 to 240±44 pmol³²P/mg MF protein) without consistent changes in phosphorylation of C-protein. In sarcoplasmic reticulum membrane vesicles, no phospholamban phosphorylation was detected either by -adrenergic stimulation of superfused hearts or when phosphorylation conditions were optimized by direct treatment of the vesicles with cAMP-dependent protein kinase (PKA) and [y ³²P] ATP.The effect of -adrenergic stimulation on ventricular strips was studied at 30 and 22°C. At 30°C, the effects of 10^–5 to 10^–4M phenylephrine on myocardial contraction and relaxation were diminished to non significant levels by addition of propranolol. At 22°C, blockage with propranolol left a remanent positive inotropic effect (10% of the total effect of phenylephrine) and changed the phenylephrine-induced positive lusitropic effect into a negative lusitropic action. These propranolol-resistant effects were abolished by prazosin. Our results suggest that in amphibian heart, both the inotropic and lusitropic responses to catecholamines are mainly due to a -adrenergic stimulation which predominates over the -adrenergic response. Phospholamban phosphorylation seems not to be involved in mediating the positive lusitropic effect of -adrenergic agents whereas phosphorylation of troponin 1 may play a critical role.     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.