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)

We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.
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PMID:Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. 1177 82

Glycohydrolases are a group of enzymes contained predominantly within lysosomes, which are released during Kupffer cell activation or death. One of these, beta-galactosidase, has been proposed as a marker of ischemia-reperfusion injury in the liver because Kupffer cell activation represents a primary event in the injurious reperfusion cascade. In this study, we compared B-galactosidase with more traditional indicators of liver injury and function in a porcine model of liver preservation. Porcine livers were allocated into two groups: group C (n = 5), preserved in University of Wisconsin solution by standard cold storage for 24 hours, and group W (n = 5), perfused with oxygenated autologous blood on an extracorporeal circuit for 24 hours. Both groups were subsequently tested on the circuit during a 24-hour reperfusion phase. The perfusate was sampled for levels of beta-galactosidase, as well as traditional markers of liver injury and function. A sharp increase in beta-galactosidase levels was seen on reperfusion of cold preserved livers to a level of 1,900 IU/mL. This contrasted dramatically with normothermically preserved livers, in which the level never exceeded 208 IU/mL (P =.002). beta-Galactosidase levels showed much earlier and greater increases compared with transaminase levels in livers injured by ischemia. A rapid elevation in beta-galactosidase levels corresponded well with poor liver function and more liver injury. Measurement of beta-galactosidase is a simple test that quantifies ischemia-reperfusion injury of preserved livers. It is more sensitive than transaminases, with faster and larger increases in levels after ischemic injury. It can be useful in assessing the viability of a liver during machine preservation.
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PMID:Beta-galactosidase as a marker of ischemic injury and a mechanism for viability assessment in porcine liver transplantation. 1179 81

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 single copy of a reporter gene cassette, such as PGKP-lacZ-LEU2 (promoter-reporter-marker gene) cassette, was inserted into one of 32 positions along chromosome III in Saccharomyces cerevisiae with an interval of approximately 10 kb. The amounts of translational gene product (beta-galactosidase) synthesized by the cassette-transformed cells were then determined. The region specificity in chromosome III could be demonstrated in gene expression: two higher-expressed regions (hot regions), 133 and 199 (MAT) regions, and seven lower-expressed regions (cold regions). For the steady and high production of polypeptide, foreign gene products, by yeast, we would like to state that we hope for an insertion of the artificially prepared gene cassette [(promoter)-(foreign gene)-(marker gene) ] into a hot region, such as 199 (MAT) region of chromosome III.
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PMID:Region specificity of chromosome III on gene expression in the yeast Saccharomyces cerevisiae. 1250 22

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.
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PMID:Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase. 1291 15

The potential of 89 culturable cold-adapted isolates from uncontaminated habitats, including 61 bacterial and 28 yeast strains, to utilize representative fractions of petroleum hydrocarbons (n-alkanes, monoaromatic and polycyclic aromatic hydrocarbons) for growth and to produce various enzymes at 10 degrees C was investigated. The efficiency of bacterial and yeast strains was compared. The growth temperature range of the yeast strains was significantly smaller than that of the bacterial strains. Sixty percent of the yeasts but only 8% of the bacteria could be classified as true psychrophiles, showing no growth above 20 degrees C. A high percentage (89%) of the yeast strains showed lipase activity. More than one-third of the 61 bacterial strains produced amylase, beta-lactamase, beta-galactosidase or lipase; more than two-thirds were protease producers. Only 6% of the bacterial strains but 79% of the yeast strains utilized n-hexadecane for growth; 13% of the bacterial strains and 21-32% of the yeast strains utilized phenol, phenanthrene or anthracene for growth. Only four yeast strains but none of the bacterial strains could grow with all hydrocarbons tested. The biodegradation of phenol was investigated in fed-batch cultures at 10 degrees C. Three yeast strains degraded phenol concentrations as high as 10 mM (one strain) or 12.5 mM (two strains). Of eight bacterial strains, two strains degraded up to 10 mM phenol. The optimum temperature for phenol degradation was 20 degrees C for all eight bacterial strains and for two yeast strains. Biodegradation by five yeast strains was optimal at 10 degrees C and faster at 1 degrees C than at 20 degrees C. All phenol-degrading strains produced catechol 1,2 dioxygenase activity.
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PMID:Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts. 1294 49

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.
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PMID:Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate. 1294 99

Strong evidence suggests that replicative senescence is involved in vivo because senescent cells have been detected in human tissues associated with physiological and pathological aging processes. Chronic allograft nephropathy (CAN) appears to be a major determinant of long-term survival in kidney transplantation. Several mechanisms are potentially involved; the aim of this study was to assess the impact of replicative senescence in CAN. Replicative senescent cells were detected on renal tissue cryosection using expression of a specific marker, senescence-associated beta-galactosidase (SA-beta-Gal) at pH 6. A total of 80 frozen renal samples (67 cases of CAN and 13 controls) were studied. To validate this marker, we measured in situ telomere length in cells expressing or not expressing SA-beta-Gal using a validated quantitative fluorescence in situ hybridization technique. The presence of senescent cells was correlated with clinicopathologic data. Telomere length was significantly lower in cells expressing SA-beta-Gal than in cells that did not. Replicative senescence was present in 45 out of 67 (67%) biopsy specimens and was significantly associated with the severity of CAN. No correlation with the notion of a previous episode of acute tubular necrosis, acute rejection, extrarenal epuration, duration of cold ischemia, and the delay between transplantation and biopsy was observed. However, the age of the donor, but not that of the recipient, was correlated with the occurrence of senescent cells. These results suggest that replicative senescence is a mechanism that might be involved in the development of CAN. The age of the donor appears to be the major determinant factor in replicative senescence.
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PMID:The role of replicative senescence in chronic allograft nephropathy. 1456 89

The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.
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PMID:Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157:H7. 1471 46

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C. Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C). Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.
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PMID:Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen. 1525 24


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