Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Localization of glycosidases with naphthyl substrates

Synopsis Unsubstituted naphthyl substrates were found to be superior to substituted naphthyl, indolyl and hydroxyquinoline substrates for the histochemical demonstration of -mannosidase, -galactosidase, hetero--glycosidase, glucoamylase and sucrase-isomaltase, equivalent for -N-acetylglucosaminidase and lactase--glucosidase, and inferior for -glucuronidase and acid -galactosidase. Aldehyde fixation is necessary for the localization of lysosomal glycosidases with naphthyl substrates.i-naphthyl substrates are suitable for the detection of acid glycosidases in lysosomes and hetero--glycosidase in the cytoplasm of animal cells, and 2-naphthyl substrates can be employed for the demonstration of microvillous glycosidases and for the evaluation of the total activity of soluble glycosidases with semipermeable membranes. When naphthyl substrates are used coupling should be carried out simultaneously and hexazotized pararosaniline is the coupling reagent of choice.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Isolation of a β-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei

The extracellular -glucosidase from the filamentous fungus Trichoderma reesei QM 9414 is mainly bound to the cell wall of the fungus and only partially released into the medium. Isolation of the cell walls and its hydrolysis by enzymatic treatment with Aspergillus niger cellulase released -glucosidase, which appeared tightly associated with a cell wall polysaccharide. This polysaccharide was purified by gel filtration and ion exchange chromatography and was shown to consist of mannose, galactose, glucose, galacturonic acid and glucuronic acid. It was devoid of protein and phosphate. It reassociated both with extracellular -glucosidase as well as -glucosidase released from the fungus' cell wall. Addition of the polysaccharide to the -glucosidase in vitro increased the enzyme's activity against 4-nitrophenyl--glucoside twofold. These findings suggest, that the isolated polysaccharide functions as an anchor glycan for the -glucosidase in Trichoderma reesei.     

Anomeric preference of glucose utilization in rat erythrocytes

The -anomer of glucose relative to the -anomer was more rapidly metabolized into lactate by rat erythrocytes at 37° C (/ ratio = ca. 1.3): the amounts of - and -D-glucose metabolized into lactate during 3 min were 0.21 and 0.27 mol/gHb, respectively. Also, the transport of -D-glucose into erythrocytes was more rapid than that of -D-glucose: the amounts of - and -D-glucose transported into erythrocytes during 3 min were approximately 3.5 and 5.0 mol/gHb, respectively. Glucose phosphorylation by rat erythrocyte hexokinase (i.e., a possible rate-limiting step in glycolysis) occurred at higher velocities with the -anomer than with the a-anomer (/ ratio = 1.28). The Km value of hexokinase for either anomer of glucose was 53 M. The glucose concentrations in erythrocytes incubated with - and -D-glucose reached about 1 mM in 1 min, indicating that hexokinase is almost completely saturated with glucose within less than 1 min. The results suggest that glucose phosphorylation and glucose transport are major and minor determinants, respectively, for the anomeric preference of glucose utilization in rat erythrocytes.     

Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of -galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) with 5-bromoindolyl--o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial -galactosidase (lacZ). After -galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for -galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of -galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

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.