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)

Commercially obtained E. coli beta-galactosidase was stored at 25 degrees C in buffer containing 1 mM MgCl2 and in buffer containing no added MgCl2. Samples were removed at set times and the activity of individual enzyme molecules assayed. When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period. When stored in the absence of added MgCl2, over half the enzyme molecules became inactive within the first hour. However, those molecules which retained activity remained active for the duration of the experiment. This indicates that there may exist two populations of E. coli beta-galactosidase, one which requires storage in the presence of the higher concentration of Mg2+ in order to remain active. There was no observed correlation between this requirement for magnesium and reaction rate. Additionally, the presence of the 1 mM MgCl2 was found to decrease the average activity of the beta-galactosidase molecules under the conditions employed.
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PMID:Escherichia coli beta-galactosidase is heterogeneous with respect to a requirement for magnesium. 1112 94

The enzyme beta-galactosidase was purified from a cold-adapted organism isolated from Antarctica. The organism was identified as a psychotrophic Pseudoalteromonas sp. The enzyme was purified with high yields by a rapid purification scheme involving extraction in an aqueous two-phase system followed by hydrophobic interaction chromatography and ultrafiltration. The beta-galactosidase was optimally active at pH 9 and at 26 degrees C when assayed with o-nitrophenyl-beta-D-galactopyranoside as substrate for 2 min. The enzyme activity was highly sensitive to temperature above 30 degrees C and was undetectable at 40 degrees C. The cations Na+, K+, Mg2+ and Mn2+ activated the enzyme while Ca2+, Hg2+, Cu2+ and Zn2+ inhibited activity. The shelf life of the pure enzyme at 4 degrees C was significantly enhanced in the presence of 0.1% (w/v) polyethyleneimine. The pure beta-galactosidase was also evaluated for lactose hydrolysis. More than 50% lactose hydrolysis was achieved in 8 h in buffer at an enzyme concentration of 1 U/ml, and was increased to 70% in the presence of 0.1% (w/v) polyethyleneimine. The extent of lactose hydrolysis was 40-50% in milk. The enzyme could be immobilized to Sepharose via different chemistries with 60-70% retention of activity. The immobilized enzyme was more stable and its ability to hydrolyze lactose was similar to that of the soluble enzyme.
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PMID:Beta-galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis. 1193 82

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.
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PMID:Purification and characterization of a beta-galactosidase from peach (Prunus persica). 1266 63

Bragg, P. D. (University of British Columbia, Vancouver, Canada) and W. J. Polglase. Action of dihydrostreptomycin and antagonism by cations. J. Bacteriol. 85:590-594. 1963.-A number of antibiotics including dihydrostreptomycin inhibited the induction of beta-galactosidase in Escherichia coli and, except in the case of chloramphenicol, magnesium antagonized the inhibition. Of the antibiotics tested, only dihydrostreptomycin caused formation of pyruvate from the oxidation of glucose. Under similar conditions, inhibitors of terminal respiration (cyanide, azide, amobarbital) also caused formation of pyruvate. Magnesium and the polyamines, putrescine and spermidine, were observed to antagonize dihydrostreptomycin in systems in which the antibiotic showed an inhibitory action.
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PMID:ACTION OF DIHYDROSTREPTOMYCIN AND ANTAGONISM BY CATIONS. 1404 36

We report that neurotrophin-3 (NT-3), delivered chronically via fibroblasts implanted intrathecally into neonatal rats, can facilitate synaptic transmission in the spinal cord. A small collagen plug containing NT-3-secreting fibroblasts was placed on the exposed dorsal surface of the spinal cord (L1) of 2-d-old rats; controls received beta-galactosidase-secreting fibroblasts. After 6 hr to 12 d of survival, synaptic potentials (EPSP) elicited by two synaptic inputs, L5 dorsal root and ventrolateral funiculus (VLF), were recorded intracellularly in L5 motoneurons in vitro. Preparations treated with NT-3 implants exhibited enhanced monosynaptic synaptic transmission from both inputs, which persisted over the entire testing period. Unlike acute enhancement of transmission by NT-3 (Arvanian and Mendell, 2001a), the chronic effect could occur at connections not normally eliciting an NMDA receptor-mediated response at the time of NT-3 exposure. Using susceptibility to blockade of the NMDA receptor by Mg2+ and APV, we confirmed that chronic treatment with NT-3 did not enhance NMDA receptor activity at these connections. Cords treated with chronic NT-3 also transiently displayed polysynaptic components activated by VLF that were blocked by the NMDA receptor antagonist APV. These novel NMDA receptor-mediated potentials may reflect changes in interneurons near the site of fibroblast implantation. We conclude that chronic NT-3 enhances the potency of segmental and descending projections via mechanisms different from those underlying acute changes.
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PMID:Chronic neurotrophin-3 strengthens synaptic connections to motoneurons in the neonatal rat. 1450 70

C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred. After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.
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PMID:Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration. 1475 8

N-methyl-D-aspartate (NMDA) responsiveness of motoneurons declines during the initial 2 postnatal weeks due to increasing Mg2+ block of NMDA receptors. Using gene chip analyses, RT-PCR, and immunochemistry, we have shown that the NR2D subunit of the NMDA receptor (NMDAR), known to confer resistance to Mg2+ block, also declines in motoneurons during this period. We injected a viral construct (HSVnr2d) into the lumbar spinal cord on postnatal day 2 in an attempt to restore NMDAR function in motoneurons during the second postnatal week. Following HSVnr2d injection, we detected elevated levels of NR2D mRNA in spinal cord samples and NR2D protein specifically in motoneurons. These molecular changes were associated with marked functional alterations whereby NMDAR-mediated responses in motoneurons associated with both dorsal root (DR) and ventrolateral funiculus (VLF) inputs returned to values observed at E18 due to decreased Mg2+ blockade. Viruses carrying the beta-galactosidase gene did not induce these effects. NT-3 is known to potentiate AMPA-kainate responses in motoneurons if the response has an NMDAR-mediated component and thus is normally ineffective during the second postnatal week. Restoration of NMDAR-mediated responsiveness in the second postnatal week was accompanied by a return of the ability of neurotrophin-3 (NT-3) to potentiate the AMPA-kainate responses produced by both DR and VLF synaptic inputs. We conclude that delivery of the gene for a specific NMDA subunit can restore properties characteristic of younger animals to spinal cord motoneurons. This approach might be useful for enhancing the function of fibers surviving in the damaged spinal cord.
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PMID:Viral delivery of NR2D subunits reduces Mg2+ block of NMDA receptor and restores NT-3-induced potentiation of AMPA-kainate responses in maturing rat motoneurons. 1538 39

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.
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PMID:Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 beta-galactosidases. 1623 52

In layer 4 of the somatosensory cortex, the glutamatergic synapses that interconnect spiny stellate (SpS) neurons, which are the major targets of thalamocortical input, differ from most other neocortical excitatory synapses in that they have an extremely large NMDA receptor (NMDAR)-mediated component that is relatively insensitive to voltage-dependent Mg2+ blockade. We now report that this unique feature of the NMDA response reflects the distinctive subunit composition of the underlying receptors. We studied NMDAR-mediated miniature EPSCs (mEPSCs) and NMDA channel currents in tangential brain slices of mouse barrel cortex, which exclusively contain layer 4. NMDAR-mediated mEPSCs in SpS neurons were prominent at negative membrane potentials, and NMDA channels in outside-out patches excised from the somata of the same neurons had relatively low conductance and reduced susceptibility to Mg2+ block. These are characteristic features of heteromeric NMDAR assemblies that contain the NR2C subunit. Some patches also contained NMDA channels with higher conductance and a greater sensitivity to Mg2+. In the neocortex of transgenic mice in which a beta-galactosidase (lacZ) indicator gene was controlled by the NR2C promoter, the lacZ indicator was densely expressed in layer 4. In current-clamp recordings, blockade of NMDARs caused hyperpolarization and an increase in apparent input resistance. Our data demonstrate that the SpS neurons of layer 4 functionally express NR2C subunits; this is the likely explanation for their ability to generate large NMDAR-mediated EPSPs that are effective at resting potential, without previous depolarization.
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PMID:NMDA receptors in layer 4 spiny stellate cells of the mouse barrel cortex contain the NR2C subunit. 1640 68

RNA loop-loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop-loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na+ are equivalent to those in the presence of near physiological Mg2+ concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative beta-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5' A(A)/(G)N6A 3'), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization.
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PMID:Stabilities of HIV-1 DIS type RNA loop-loop interactions in vitro and in vivo. 1641 Jun 13


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