Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya- crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.
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PMID:Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP. 749 74

The product yield of staphylococcal Protein A reached only 1.8% of the cell dry weight, while the corresponding value was 14% for a fusion protein composed of Protein A and Escherichia coli beta-galactosidase [1], when produced in the same E. coli host strain, with the same promoter and under identical process conditions. Measurement of the stability of Protein A in vivo showed that it was quickly degraded in the cell with a half-life of 30 min when the protein was expressed alone, but after fusion to beta-galactosidase, the Protein A part became considerably stabilized. In spite of the fast intracellular proteolysis of Protein A, few degradation products could be identified on Coomassie Brilliant Blue-stained SDS/PAGE gels after IgG purification, indicating an even faster degradation of the Protein A fragments. Such degradation products, however, accumulated during incubation of the disintegrated cells. Intracellular degradation intermediates could be demonstrated with the more sensitive Western-blot technique. This technique also revealed that a slow degradation took place not only in the Protein A moiety of the fusion protein, but also in the beta-galactosidase moiety. A control with native beta-galactosidase also showed a weak in vivo proteolysis of this molecule, but it was more stable in free form than in the fused form. This means that the proteolytically very sensitive Protein A was stabilized by fusion with beta-galactosidase, but the originally rather stable beta-galactosidase became slightly more susceptible to proteolysis after the fusion.
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PMID:Proteolysis of fusion proteins: stabilization and destabilization of staphylococcal protein A and Escherichia coli beta-galactosidase. 757 56

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.
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PMID:Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons. 767 15

Increased activity of the cellular Na(+)-H+ exchanger (NHE) has been documented in various cell types in essential hypertension and in vascular myocytes of the spontaneously hypertensive rat (SHR). The mechanism underlying this abnormality is unclear. Because the NHE can be activated by phosphorylation, we examined phosphorylation of the Na(+)-H+ exchanger isoform 1 (NHE-1) as one possible mechanism for its increased turnover number in cultured vascular myocytes of the SHR. A polyclonal rabbit antibody against a fusion protein consisting of beta-galactosidase and the C-terminus of NHE-1 was used to immunoprecipitate 32P-labeled NHE-1 from cell extracts of SHR and Wistar-Kyoto (WKY) rat vascular myocytes in the absence and presence of 10% fetal calf serum. Immunoprecipitates were separated by SDS-PAGE, and 32P-labeled NHE-1 was quantified from autoradiographs. Similar amounts of NHE-1 protein were detected on Western blots of the cultured vascular myocytes from SHR and WKY rats. In quiescent cells, NHE-1 was significantly more phosphorylated in SHR myocytes than in WKY myocytes (2.17 +/- 0.06-fold enhancement [mean +/- SEM]; P < .001, n = 8). The addition of fetal calf serum to quiescent cells had no significant effect on the phosphorylation of NHE-1 in SHR myocytes. However, NHE-1 phosphorylation fell transiently in serum-treated WKY myocytes, with recovery to control levels after 20 minutes. Measurement of NHE activity using fluorometry confirmed elevated activity in the quiescent SHR myocytes compared with WKY myocytes. Fetal calf serum led to further enhancement of NHE activity in both cell types. These findings suggest that the increased NHE activity in quiescent SHR myocytes may be correlated with enhanced NHE-1 phosphorylation and that serum stimulates NHE activity in both cell types without a further increase in total NHE-1 phosphorylation, indicating a role for non-phosphorylation-dependent regulatory mechanisms.
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PMID:Na(+)-H+ exchanger isoform 1 phosphorylation in normal Wistar-Kyoto and spontaneously hypertensive rats. 772 99

The receptor protein for thyrotrophin (thyroid-stimulating hormone; TSH) is associated with a glycosphingolipid moiety. The protein belongs to the family of receptors that couple to guanine nucleotide binding proteins; the glycosphingolipid contains sialic acid and belongs to the family of gangliosides. This report defines the structure of the receptor ganglioside in the Fisher rat thyroid cell line (FRTL-5). Receptor protein was purified by TSH affinity chromatography from FRTL-5 cells, biosynthetically labelled with [3H]galactose and [3H]glucosamine, and resolved by SDS-PAGE. A single radiolabelled band of Mr approximately 80 kDa, corresponding to the predicted size of the cloned receptor, contained ganglioside. Gangliosides were extracted from unlabelled receptor protein after SDS-PAGE and probed on TLC plates with 125I-labelled Limax flavus agglutinin or the B subunit of cholera toxin, before and after digestion with Vibrio cholerae sialidase or beta-galactosidase. The TSH receptor (TSH-R) ganglioside belongs to the gangliotetraose family, having sialic acid attached to both galactose molecules. Its sialic acid is devoid of negative charge because of the formation of internal esterlactones. Its structure is lactonized N-acetylneuraminyl-(alpha 2-->3)galactosyl(beta 1-->3)-N-acetylgalactosaminyl(beta 1-->4)-[N-acetylneuraminyl(alpha 2-->3)]galactosyl(beta 1-->4)glucosyl(beta 1-->1)ceramide (GDla-lactone). Ganglioside lactones have not been previously described as components of thyroid cells. They are highly rigid and are more likely than their parent structures to serve as molecular recognition sites and elicit immunoreactivity. Identification of this unique ganglioside intimately associated with the TSH receptor implies that it has an integral role in receptor structure and function.
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PMID:Characterization of ganglioside associated with the thyrotrophin receptor. 773 42

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a beta-galactosidase gene. The transformants expressed a constitutive beta-galactosidase activity at a higher level than in Lact. casei, and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and beta-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.
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PMID:Expression of Lactobacillus casei ATCC 393 beta-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123. 776 47

The baculovirus infection process of Spodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinant Autographa californica (AcNPV) virus expressing beta-galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique--SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG)--was used to monitor beta-galactosidase production. Specific beta-galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units beta-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.
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PMID:Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells. 776 30

An exo-(1-->3)-beta-D-galactanase was purified by six chromatographic steps from a culture supernatant of Aspergillus niger. Its apparent molecular mass was 66 kDa, as estimated by SDS-PAGE analysis. The purified enzyme had no detectable activity on various p-nitrophenyl glycosides and on native plant polysaccharides but exhibited a high activity on a (1-->3)-beta-D-linked galactan backbone obtained after partial acid hydrolysis and two Smith degradations of gum arabic. The optimum conditions were pH 4.5 and 40-50 degrees C. The enzyme had a Michaelis constant (Km) of 1.9 mg/mL for the beta-(1-->3)-D-galactan with a maximum reaction velocity (Vmax) of 1380 nkat/mg. The study of the reaction products obtained after enzyme treatment of two galactans derived from gum arabic through one or two Smith degradations showed that it was an exo-(1-->3)-beta-D-galactanase able to by-pass the branching points of galactan backbones and thus to release the side-chains of type II arabinogalactans in an undegraded form.
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PMID:Purification and properties of an exo-(1-->3)-beta-D-galactanase from Aspergillus niger. 780 66

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.
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PMID:Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase. 781 96

Serum resistance of gonococci in most patients is due to sialylation of a Gal beta 1-4GlcNAc group on a conserved 4.5 kDa lipopolysaccharide (LPS) component by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) catalysed by a gonococcal sialyl transferase. This sialylation is enhanced by a low M(r) factor(s) which, like CMP-NANA, is released in diffusates from high M(r) fractions obtained from sonicates dialysed at 4 degrees C. Also, as shown here, this factor(s) is released when the sonicates are dialysed at 18-20 degrees C. The enhancement of sialylation, first demonstrated using enzymes in gonococcal extracts, has been shown to occur in live gonococci and hence probably to have a role in pathogenicity. Gonococci, emerging from lag phase and incubated for 2 h with CMP-14CNANA fixed up to 90% more radiolabel than controls when the second factor(s) was present; their LPS separated by SDS-PAGE contained more radiolabel than control samples and label was not detected in any other component. Fractions with enhancing activity absorbed maximally at about 260 nm but a mixture of UDP-galactose (UDP-Gal), UDP-N-Acetyl galactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc) and UDP-N-Acetyl glucosamine (UDP-GlcNAc) showed no significant enhancing activity. The enhancing action of the low M(r) fractions was unaffected by incubation with beta-galactosidase.
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PMID:Sialylation of lipopolysaccharide by CMP-NANA in viable gonococci is enhanced by low Mr material released from blood cell extracts but not by some UDP sugars. 783 May 28


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