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1.
Summary Strains of Clostridium acetobutylicum were tested for the presence of -galactosidase and phospho--galactosidase activities when grown on lactose. All strains, except C. acetobutylicum ATCC 824, showed both enzyme activities. Only phospho--galactosidase activity was detected with C. acetobutylicum ATCC 824. C. acetobutylicum strains P262 and ATCC 824 showed no detectable -galactosidase or phospho--galactosidase activities when grown on glucose. In the fermentation of whey permeate C. acetobutylicum P262 showed an early induction of phospho--galactosidase associated with the acidogenic phase. The -galactosidase activity peaked at a later stage of the fermentation (22 h) coinciding with the solvent production phase. Similar induction of phospho--galactosidase at the early stages (13 h) of fermentation of whey permeate by C. acetobutylicum ATCC 824 was also shown. No -galactosidase activity was detected during the entire course of fermentation by strain ATCC 824.  相似文献   

2.
The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.  相似文献   

3.
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  相似文献   

4.
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.  相似文献   

5.
Summary Effects of pH on -galactosidase expression and stabilization were investigated using recombinantE. coli harboring an expression vector with a thermally-inducible PL promoter. Expression of -galactosidase was strongly promoted by lowering culture pH when culture temperature was raised to the induction temperature. Optimal pH for induction ranged from 5.4–5.8. The degradation of expressed -galactosidase could be reduced by lowering the culture temperature while at the same time slightly increasing the culture pH in the -gal degradation stage.  相似文献   

6.
Summary Hybrid cell lines isolated after fusions between Chinese hamster E36 cells and normal human white blood cells were analyzed for human -galactosidase isoenzymes and for human chromosomes, especially 3, 12, and 22, the candidates for bearing a -galactosidase locus. Results of neuraminidase treatment of the cell lysates and immunological studies showed that in man two structural -galactosidase loci are present and can be assigned to chromosomes 3 and 22. No correlation was found between the expression of human -galactosidase and the presence of human chromosome 12.  相似文献   

7.
A new approach for the highly specific preparation of L-serine conjugates of lactosamine and Gal1-3GalNAc is described. Thus, the L-serine derivative of lactosamine Gal1-4GlcNAc-O-(N-Z)-Ser-OEt, was obtained from lactose, employing GlcNAc-O-(N-Z)-Ser-OEt as acceptor and a yeast -galactosidase as catalyst Galp 1-3GalNAc-O-(N-Alloc)-Ser-OMe was obtained from lactose, employing GalNAc-O-(N-Alloc)-Ser-OMe as acceptor and -galactosidase from bovine testes as catalyst.  相似文献   

8.
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.  相似文献   

9.
The role of -galactosidase repression in glucose inhibition of lactose utilization was studied inEscherichia coli. Escherichia coli 3300 constitutively produces -galactosidase even in the presence of glucose. When this strain was grown in a mixture of glucose and lactose, lactose utilization did not occur until glucose was depleted. The addition of glucose to a 3300 culture grown in lactose immediately caused a permanent inhibition of lactose utilization and only a mild transient repression of -galactosidase. Exogenous cyclic adenosine monophosphate (AMP) did not overcome the glucose inhibition of lactose utilization but did relieve the transient repression. Thus glucose inhibition of lactose utilization is not related to -galactosidase repression and is independent of cyclic AMP.  相似文献   

10.
GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside  相似文献   

11.
Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-14C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg2+: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg2+, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca2+ dependent and this dependence could be abolished by proteinase treatment. The K m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.  相似文献   

12.
Zusammenfassung Es wird ein simultanes Azokupplungsverfahren zur intrazellulären Darstellung der sauren (Hetero-) und neutralen -Galactosidase (Lactase) in verschiedenen Organen von Ratte, Maus und Meerschweinchen beschrieben.Das Inkubationsmedium enthält 4,5–9mg 1-Naphthyl--galactopyranosid (gelöst in 0,4ml NN-Dimethylformamid) und 0,5–0,8ml 2% Hexazonium-p-rosanilin in 9 ml 0,1 M Citrat-Puffer, pH 5 (Hetero--galactosidase) oder 5,5 (Lactase).Unter allen Organen reagiert die saure -Galactosidase am kräftigsten in den Lysosomen von Nebenhoden, Niere, Nebenniere, Schilddrüse, Glandula präputialis und inguinalis, Milz, Colon und Plexus chorioideus; die neutrale -Galactosidase kommt in mittlerer Aktivität nur im intestinalen Stäbchensaum vor.Die intralysosomale Darstellung der löslichen Hetero--galactosidase erfordert Blockfixation in Glutaraldehyd; die Lactase kann an frischen oder gefriergetrockneten Schnitten untersucht werden. Im proximalen Tubulus der Rattenniere wird die saure -Galactosidase durch Formol unabhängig von der Konzentration des Fixans verglichen mit Glutaraldehyd stärker gehemmt. Spätestens 10 min nach Beginn der Fixation hat das Enzym seine Basisaktivität erreicht. Spülen in hypertoner Zuckerlösung macht die Inhibition der Hetero--galactosidase teilweise rückgängig.Die mit dem Azokupplungs- und Indigogen-Verfahren gewonnenen Befunde sind weitgehend identisch.
On the histochemical and microchemical demonstration of -galactosidase by means of 1-naphthyl--galactopyranoside
Summary A simultaneous azo coupling method for the intracellular demonstration of acid (hetero-) and neutral -galactosidase (lactase) in various organs of rats, mice and guinea-pigs is described.The recommended incubation medium consists of 4.5–9 mg 1-naphthyl--galactopyranoside (dissolved in 0.4 ml NN-dimethylformamide) and 0.5–0.8 ml 2% hexazonium-p-rosaniline in 9 ml 0.1 M citrate buffer, pH 5.0 (hetero--galactosidase) or 5.5 (lactase).Among all organs investigated the strongest acid -galactosidase reaction regularly occurs in the lysosomes of the epididymis, kidney, adrenal, thyroid, preputial and inguinal gland, spleen, colon and chorioid plexus; the neutral -galactosidase can only be detected in the intestinal brush border exhibiting a moderate activity.Because hetero--galactosidase is a highly soluble enzyme bloc-fixation using glutaraldehyde becomes necessary to achieve a precise intralysosomal localization; for the demonstration of lactase fresh or freeze-dried cryostat sections are suitable. —In the proximal tubule of the rat kidney independent of their concentration the inhibition of acid -galactosidase following treatment with formol surpasses that of glutaraldehyde. Within the first ten minutes of fixation the enzyme reaches its basis activity. The recovery rate of renal hetero--galactosidase considerably increases in the course of washing in hypertonic sugar solution.In comparison with the indigogenic technique nearly identical results can be obtained with the azo coupling procedure.
  相似文献   

13.
Summary The -galactosidase product of the DNA-directed cell-free system for the synthesis of protein of the lac operon, developed by Zubay and his colleagues, has been purified to radioactive homogeneity and compared to wild-type -galactosidase. When analyzed by sucrose gradient centrifugation, SDS-gel electrophoresis, and kinetic analysis, the purified cell-free enzyme behaves identically to the purified wild-type -galactosidase.  相似文献   

14.
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.  相似文献   

15.
Summary Chimeric -galactosidase fusion proteins containing foreign peptides inserted either at the amino terminus or at inner sites have been studied regarding their purification properties. Whereas fusions at the amino terminal are retained less on TPEG-Sepharose columns than native -galactosidase, the insertion in a specific site of the activating interface increases the binding of the modified -galactosidase. This offers a way to construct more powerful -galactosidase purification tags.  相似文献   

16.
The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)  相似文献   

17.
The properties and distribution of -galactosidase were studied in the mouse brain using the artificial substrate methylumbelliferyl--galactoside. Enzyme activities were compared between an audiogenic seizure-susceptible mouse strain (DBA/2) and three non-susceptible strains of mice (BALB/c, C3H/He and Swiss A2G). At all ages, DBA/2 mice have significantly lower -galactosidase activity compared with the three other mouse strains: this is attributed to the different alleles present at the Bgs locus. The low activity of -galactosidase is also evident when the natural substrate GMI-ganglioside is hydrolyzed. In contrast to this low GMI-ganglioside--galactosidase activity, there is no difference in the activity of the second form of acid -galactosidase, galactosylceramidase, in DBA/2 mice at 7 and 14 days. However, at 21 and 28 days the activity is significantly lower in DBA/2 mice compared with the other strains of mice. These results on -galactosidase activity in the brain of seizure-susceptible and non-susceptible mice are discussed in relation to published levels of GMI-ganglioside and galactosylceramide present in the developing mouse brain.Dedicated to Henry McIlwain.  相似文献   

18.
Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.  相似文献   

19.
Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R MTet chromatographic migration rate in relation to that of maltotetraose  相似文献   

20.
Summary Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and -galactosidase. The mutants were isolated as TonB- Lac+ derivatives of a phenotypically Lac- TrpR- strain carrying a lac I +-Z+ fusion on a 80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA- and 3 Trp- strains produce triple chimeras. The chimeras from the two TrpA- strains were further examined. They consist of tryptophan synthetase -subunit, lac repressor and -galactosidase. In crude extracts of these strains the tryptophan synthetase -subunit part can be identified by its ability to aggregate with the -subunit since some of the -subunit activity can be precipitated with antiserum against -galactosidase. Furthermore -galactosidase precipitates with antiserum against tryptophan synthetase -subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The -galactosidase part is as unaffected as in the original lac repressor--galactosidase chimera. The molecular weigths of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.  相似文献   

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