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)

The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.
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PMID:Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter. 136 36

A heterogeneous enzyme-labeled immunometric assay for quantifying digoxin in serum or plasma was developed. The sample's drug reacts with an excess of an antidigoxin Fab'-beta-galactosidase monoconjugate and then the free monoconjugate is removed by polyacrylamide digitoxigenin-coupled beads; the beta-galactosidase activity of the supernatant measured photometrically at 634 nm is directly proportional to the digoxin concentration in the sample. The assay shares the basic reagents for the immunological reaction with the Seralyzer dry-strip immunometric assay; the reagents for the indicator enzyme reaction were instead formulated in liquid form for use with common clinical analyzers. The test requires a two-point calibration (a 3.0 micrograms/L digoxin calibrator and a reagent blank). One sample can be assayed in approximately 20 min; the assay range is from 0.3 to 5.0 micrograms/L. Dilution tests showed an average found/expected ratio of 100.6%. Mean analytical recovery was 99.2%. Overall coefficients of variation (CVs) (three replicates for 12 runs over 15 days; daily calibration) ranged from 4.9 to 10.0% (Technicon RA-50) and from 2.5 to 10.0% (Cobas Fara) for samples with digoxin concentrations of 4.7 to 0.5 micrograms/L. No interference was found by high levels of common analytes (bilirubin, triglycerides, hemoglobin, total protein, uric acid) or anticoagulants. Cross-reactivity by digoxin metabolites and structurally-related compounds was investigated. Good correlations were found with radiommunoassay (RIA) (r = 0.986) and enzyme immunoassay (EIA) (r = 0.994). Thus, the assay is a specific, reliable, and convenient method for measuring digoxin in both small and large laboratories.
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PMID:An enzyme-labeled immunometric assay for quantitation of digoxin in serum or plasma. 154 93

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.
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PMID:Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent. 163 20

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.
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PMID:Identification and cloning of a fur regulatory gene in Yersinia pestis. 189 28

Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of protein-acetaldehyde adducts (-AAs) in human serum samples. Two methods were compared: (1) direct ELISA: samples, rabbit anti-hemocyanin-AA IgG, and beta-galactosidase (beta-gal) conjugated goat anti-rabbit serum IgG added to a 96-well ELISA plate in a stepwise manner; and (2) two-site or sandwich ELISA: serum samples added to an ELISA plate that had been precoated with anti-hemocyanin-AA IgG (the capture antibody) and incubated stepwise with biotinated anti-hemocyanin-AA IgG (the signal antibody) and avidin-beta-gal conjugates. Serum protein-AA levels were then assayed by bound beta-gal activities at OD405. When human hemoglobin (Hgb)-AA was used as a model protein-AA for the sandwich ELISA, the EC50 (estimated concentration that corresponds to 50% of the OD405 response range) was 7 ng/ml. Direct ELISA was less sensitive (EC50 of 120 ng/ml). Adding control human serum to Hgb-AA increased the EC50 of the direct ELISA more than the sandwich ELISA. Intra- and interassay coefficients of variance for sandwich ELISA were both about 8%. Detection of Hgb-AA by sandwich ELISA was highly specific. The above results with anti-hemocyanin-AA IgG were also obtained when anti-myoglobin-AA IgG was used in sandwich ELISA. Using sandwich ELISA and anti-hemocyanin-AA IgG, OD405 for sera of control subjects and alcoholic patients were 0.036 +/- 0.033 (+/- SEM, n = 28) and 0.150 +/- 0.088 (n = 28), respectively. Serum protein-AAs reacted more strongly with anti-myoglobin-AA IgG than anti-hemocyanin-AA IgG.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein-acetaldehyde adducts in serum of alcoholic patients. 237 29

Adler and Martin (1983, Curr. Eye Res. 2, 359-66) found cathepsin D to be present in crude preparations of bovine interphotoreceptor matrix (IPM). The purpose of the present study was to determine, by investigating several acid hydrolases in purer IPM samples, whether hydrolytic enzymes abundant in RPE lysosomes were present also as normal components of the IPM. IPM was prepared from bovine eyes by the introduction of a small bleb of buffer between the neural retina and the RPE. These IPM samples were free from significant contamination by surrounding tissues; they contained IRBP as their only major protein, and had negligible amounts of lactate dehydrogenase and ROS-specific proteins. Most acid hydrolases were assayed fluorometrically by measuring the 4-methylumbelliferone released upon hydrolysis of appropriate derivatives; the substrate for cathepsin was hemoglobin. The amounts of the enzymes found in the IPM were far from uniform and could not be correlated with enzyme activities in either RPE or retina homogenates. The hydrolases in the IPM varied in amount from beta-galactosidase (28% of the RPE level), through N-acetyl-beta-glucosaminidase (20%), alpha-fucosidase (15%), beta-glucuronidase (12%), alpha-glucosidase (8%), cathepsin D (7%), alpha-mannosidase (7%), down to beta-glucosidase, acid phosphatase, and acid lipase (trace amounts, less than 1%). These results agree with the relative amounts of enzymes found by Wilcox (1987) to be secreted into the medium by cultured human RPE cells. Furthermore, the rank order of hydrolases in the IPM is the same as that for hydrolases secreted (but not recaptured) by human fibroblasts in I-cell disease. The conclusion from these correlations is that lysosomal enzymes are probably secreted, as a normal process, by the RPE into the IPM, where they may have a role in digesting shed outer segments and in catabolizing IPM components.
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PMID:Selective presence of acid hydrolases in the interphotoreceptor matrix. 261 85

This is a kinetic assay for measuring K+ in serum, based on the activation of pyruvate kinase (EC 2.7.1.40) by K+. We eliminated interference from Na+ and NH4+ ions, which also activate this enzyme, by including Na+-binding and NH4+-consuming reagents in the reaction mixture. The assay was developed with and evaluated in the Cobas Fara centrifugal analyzer (and has been used in other kinetic analyzers). Within-run and between-run CVs were less than 1.4% and less than 1.6%, respectively. The reaction rate per millimole of K+ per liter (0.05 delta A/min) was more than double that of the reagent blank (0.02 delta A/min). Results correlated well with those by flame photometry, and interference from bilirubin, hemoglobin, lipids, heparin, and other cations was negligible. This method, in conjunction with a previous method we have reported in which beta-galactosidase is used for measuring Na+ in serum, offers a practical alternative to the use of ion-selective electrodes and flame photometry for measuring these clinically important monovalent cations in high-throughput or "stat" biochemical analyzers.
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PMID:Enzymatic determination of potassium in serum. 272 Sep 76

Urinary excretion of glycosaminoglycans (GAGS) and sialic acid (SA), as well as the activity of two renal enzymes related to glycoprotein metabolism, N-acetyl-beta-D-glucosaminidase (NAG) and beta-galactosidase (GAL), and two others unrelated to glycosaminoglycans and glycoprotein metabolism, gamma-glutamyltranspeptidase (gamma-Gt) and angiotensin-I-converting enzyme (ACE), were evaluated in 40 insulin-dependent diabetic patients with normal range albuminuria, 21 patients with mesangial glomerulonephritis, and 30 control subjects. Diabetic and glomerulonephritic patients excreted a significantly higher amount of GAGS and SA, and showed greater NAG and GAL activities; gamma-Gt and ACE levels were within normal ranges. No correlation could be demonstrated between diabetes duration and GAGS, SA, NAG and GAL findings. Moreover, no correspondence between degree of metabolic control, as reflected by glycosylated hemoglobin (HbA1a-c) and GAGS, SA, NAG and GAL emerged.
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PMID:Urinary glycosaminoglycans, sialic acid and lysosomal enzymes increase in nonalbuminuric diabetic patients. 287 16

This is a kinetic assay for measuring serum Na+ concentration based on determination of Na+-dependent beta-galactosidase (EC 3.2.1.23) activity. The method, sufficiently sensitive to measure sub-millimolar concentrations of Na+, was modified by including a Na+-binding agent (cryptand) to provide a linear assay for serum Na+ concentrations between 110 and 160 mmol/L. The assay was developed with and evaluated in the Cobas Fara centrifugal analyzer (and has been used in other kinetic analyzers). Within-run and between-run CVs were less than 1%. The reaction rate for normal serum samples (0.20 delta A/min) is about 10-fold that of the reagent blank. Results correlated well with flame photometry. Interference from bilirubin, hemoglobin, lipemia, heparin, and other cations was negligible. The method offers a practical alternative to the use of ion-selective electrodes and flame photometry for measuring serum Na+ in high-throughput or "stat" biochemical analyzers.
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PMID:Enzymatic determination of sodium in serum. 314 Oct 85

We compared the abilities of young and senescent fibroblasts to take up and degrade [3H]ribonuclease A (native and oxidized), [3H]ribonuclease4-13, [3H]hemoglobin, [3H]glyceraldehyde-3-phosphate dehydrogenase, [3H]beta-galactosidase, [3H]glycogen phosphorylase, and [125I]serum albumin. The endocytic uptake of these proteins ranged from fluid-phase to predominantly absorptive. Intralysosomal degradation rates of the different endocytosed proteins varied by an order of magnitude, but in no case was there a difference between cultures of young and senescent fibroblasts.
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PMID:Degradation of endocytosed proteins is unaltered in senescent human fibroblasts. 314 44


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