Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or -chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of -globulin hydrolyzed by trypsin or -chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed -globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with -globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of -globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Control of formation of active soluble or inactive insoluble baker''s yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

Summary Using standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast -glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated -glucosidase granules in Escherichia coli. Under these conditions active soluble -glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active -glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacI ^qrepressor gene on an R-plasmid. The formation of active soluble -glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - RB refractile body - t-PA tissue type plasminogen activator - IFN interferon - X--glucoside 5-bromo-4-chloro-indolyl--D-glucopyranoside     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Mating reaction in Saccharomyces cerevisiae

A diffusible sex-specific substance called substance-I (S-I) was isolated from culture filtrate of type strains of the yeast Saccharomyces cerevisiae. The isolated S-I, an oligopeptide, induced sexual cell agglutinability in inducible a type strains and enhanced the agglutinability in constitutive a type strains. The induction of sexual agglutinability was detected in 30 min and reached maximum in 90 min, when 0.2 g/ml of S-I was added to inducible a type cells. The a type-specific factor responsible for sexual cell agglutination, called a type agglutination factor (aAF), was shown to be produced during the induction or the enhancement of agglutinability of a type cells by S-I. The aAF produced in response to S-I was not different in the susceptibility to proteolytic enzymes and disulfide-cleaving agents from those produced constitutively in the absence of S-I.     

Symplasmic and apoplasmic radial ion transport in plant roots

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of -glucan phosphorylase were visible. Using specific -glucans incorporated into the gel before electrophoresis we have tentatively correlated -amylase and -amylase with both hydrolytic activities. The activities of -glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of -glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and -glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DDT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G1P glucose 1-phosphate - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - P_i inorganic orthophosphate - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase, EC 4.1.1.39     

Extracellular and mycelial amylases of the thermophilic fungus Malbranchea sulfurea

The thermophilic fungus Malbranchea sulfurea produces extracellular -amylase whereas -glucosidase is mainly cell bound. Extraction of the cell bound enzyme was maximum with one molar NaCl, followed by Triton ×100 and Urea-Na₂SO₃ extractants. Supplementation of Triton ×100 in growth medium significantly affected the presence of enzymes at various locations. A role for cell bound -amylase and -glucosidase has been suggested in rapid starch utilization by the fungus during early growth phase.     

Identification of a locality in snake venomα-neurotoxins with a singificant compositional similarity to marine snailα-conotoxins: Implications for evolution and structure/activity

Summary -neurotoxins from elapid snake venoms and-conotoxins from marine snails bind specifically and with high affinity to nicotinic cholinoceptors. Although both types of toxin are polypeptides, there is more than a fourfold difference in size between the two and no clear sequence homology is evident. A systematic computer search of the three-dimensional structure of erabutoxin b (an-neurotoxin from the false sea snakeLaticauda semifasciata) was performed to identify the locality that most closely matched the amino acid compositions of the smaller-conotoxins (from the marine snailsConus magus andConus geographus). The area of greatest similarity centered on residue position 25 of erabutoxin b, a locale that is conserved throughout the snake-neurotoxins and their homologues. Six Proteins unrelated to erabutoxin b were compared to the-conotoxins to show that the extent of the erabutoxin b/-conotoxin match was too high to be coincidental. Homologues of erabutoxin b, namely-cobratoxin fromNaja naja siamensis and cytotoxin V^II4 fromNaja mossambica mossambica, were also analyzed. The extent of the matching with the-conotoxins decreased in the series erabutoxin b>-cobratoxin>cytotoxin V^II4, and this also relates the order of similarity to the pharmacological properties of the-conotoxins.The-conotoxin-like area of the snake-neurotoxins is peripheral to the site previously considered important for binding to the cholinoceptor, even though it seems to represent the focus of evolutionary convergence between the two types of neurotoxin. The area of resemblance does, however, have strong associations with the conformational behavior of the snake toxins. Hence, the outcome of this study has important consequences for the current ideas on snake-neurotoxin structure/activity relationships and the evolutionary origins of neurotoxicity.