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)

A neutral beta-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A--agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with alpha-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral beta-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral beta-galactosidase during sexual maturity of epididymis in vivo.
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PMID:Purification and characterization of rat epididymal neutral beta-galactosidase and its changes during in vivo development. 832 73

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, DNA-binding, and nuclear localization activities. Deletion analysis, as well as fusions to the DNA-binding domain of GAL4, indicated that the activation potential of Egr-1 is distributed over an extensive serine/threonine-rich N-terminal domain. In addition, a novel negative regulatory function has been precisely mapped 5' of the zinc fingers: amino acids 281 to 314 are sufficient to confer the ability to repress transcription on a heterologous DNA-binding domain. Specific DNA-binding activity was shown to reside in the three zinc fingers of Egr-1, as predicted by homology to other known DNA-binding proteins. Finally, nuclear localization of Egr-1 is specified by signals in the DNA-binding domain and basic flanking sequences, as determined by subcellular fractionation and indirect immunofluorescence. Basic residues 315 to 330 confer partial nuclear localization on the bacterial protein beta-galactosidase. A bipartite signal consisting of this basic region in conjunction with either the second or third zinc finger, but not the first, suffices to target beta-galactosidase exclusively to the nucleus. Our work shows that Egr-1 is a functionally complex protein and suggests that it may play different roles in the diverse settings in which it is induced.
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PMID:A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1. 833 1

Electroelution of protein bands resolved by nondenaturing polyacrylamide gel electrophoresis was performed to identify an endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, through its biological activity. After electrophoresis protein bands were negatively stained with zinc acetate, indicating that this staining technique can be also applied to nondenaturing polyacrylamide gels. However, even under appropriate electroelution conditions, a strong fucosyl-transferase inhibitory activity was eluted from the polyacrylamide gel itself that impeded the measure of fuctinin activity. As an alternative to electroelution, collection of proteins resolved by nondenaturing polyacrylamide gel electrophoresis as they are electrophoresed off the end of the gel was assayed. In the commercially available preparative electrophoretic systems, an elution chamber is limited by a semipermeable membrane on which proteins were adsorbed in the low-ionic-strength buffer used in nondenaturing electrophoresis. We demonstrate that the simple and inexpensive electrofractionation system described by Shain et al. (Anal. Biochem. 200, 47-51, 1992) can be successfully applied under nondenaturing conditions to purify and identify fuctinin through its biological activity. However, caution must be taken in the design of the apparatus in order to avoid local heating and thermal denaturation of proteins. The utility of this nondenaturing preparative electrophoresis system for the study of functional proteins is also demonstrated by the recovery of enzymatic activities of alkaline phosphatase and beta-galactosidase.
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PMID:A nondenaturing preparative gel electrophoresis system for the recovery of functional proteins. Application to the identification of an endogenous protein inhibitor of fucosyl-transferase activities. 836 98

NRE, the nitrogen regulatory protein of Penicillium chrysogenum, contains a single Cys2/Cys2-type zinc-finger motif followed immediately by a highly basic region. The zinc-finger domain was expressed to Escherichia coli as a fusion protein with beta-galactosidase. In order to test the putative DNA-binding ability of NRE, the intergenic promoter region of the nitrate reductase/nitrite reductase gene cluster (niiA-niaD) of Penicillium was sequenced. Our results show that NRE is a DNA-binding protein and binds to the intergenic promoter regions of the P. chrysogenum niiA-niaD and acvA-pcbC gene cluster, encoding the first two enzymes in penicillin biosynthesis. Three of the four high-affinity NRE-binding sites contained two GATA core elements. In one of the recognition sites for NRE, one GATA motif was replaced by GATT. The two GATA elements showed all possible orientations, head-to-head, head-to-tail and tail-to-tail, and were separated by between 4 and 27 bp. Missing-contact analysis showed that all three purines in both of the GATA core sequences and the single adenine residue in each of the complementary TATC sequences were involved in the binding of NRE. Moreover, loss of purines in the flanking regions of the GATA elements also affect binding of NRE, as their loss causes reduced affinity.
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PMID:NRE, the major nitrogen regulatory protein of Penicillium chrysogenum, binds specifically to elements in the intergenic promoter regions of nitrate assimilation and penicillin biosynthetic gene clusters. 859 Apr 70

6-Phospho-beta-galactosidase from Lactobacillus gasseri JCM 1031 was purified from lactobacilli to homogeneity, about 118-fold, with 0.5% recovery by several chromatographies. The molecular mass and isoelectric point of the purified enzyme were 58 kDa and pI 5.5, respectively. The Km and Vmax for o-nitrophenyl-beta-D-galactopyranoside-6-phosphate were 0.8 mM and 116.4 mumol/min/mg, respectively. Reducing agent, Fe2+ ion, and EDTA activated but PCMB, Zn2+, and Hg2+ ions strongly inhibited the enzymatic activity.
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PMID:Purification and characterization of 6-phospho-beta-galactosidase from Lactobacillus gasseri JCM 1031. 882 35

In a search for genes involved in regulation of the 2'-N-acetyltransferase in Providencia stuartii, a mini-Tn5Cm insertion has been isolated in a locus designated aarD. The aarD1::mini-Tn5Cm mutation resulted in a 4.7-fold increase in the levels of beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion and a 32-fold increase in the levels of gentamicin resistance in P. stuartii. The wild-type aarD locus was cloned on a 5.0 kb Cla I fragment and complemented the aarD1 mutation. Nucleotide sequence analysis of this fragment identified two large open reading frames whose deduced products displayed significant amino acid identity, 64% and 64%, respectively, to the CydD and CydC proteins of Escherichia coli, which are involved in formation of the cytochrome d oxidase complex. Physical mapping indicated the aarD1::mini-Tn5Cm insertion was within the open reading homologous to CydD. The strain containing the aarD1 mutation was unable to grow in the presence of toluidine blue or on glycerol minimal media in the presence of zinc, suggesting that aarD is functionally equivalent to cydD. Additional phenotypes resulting from the aarD1 mutation included: altered cell morphology, a reduced growth rate and the inability of cells to grow beyond early log phase. Further examination of this phenomenon revealed that the aarD1 mutant was unable to grow in the presence of a self-produced extracellular factor(s). This novel phenotype was limited to P. stuartii as E. coli cydD and delta cydAB::kan mutants were also sensitive to a self-produced extracellular factor.
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PMID:aarD, a Providencia stuartii homologue of cydD: role in 2'-N-acetyltransferase expression, cell morphology and growth in the presence of an extracellular factor. 883 Feb 42

To study the regulation of expression of the metallothionein gene in normal liver cells, we transfected chick embryo liver cells in primary cultures with constructs containing luciferase or chloramphenicol acetyl transferase (as reporter genes) under the control of differing lengths of the 5'-promoter region of the chick metallothionein gene (containing 30, 122, 190, or 623 base pairs upstream of the transcriptional start site). We controlled for efficiency of transfection by co-transfections with a plasmid containing a bacterial beta-galactosidase gene under the control of the SV 40 promoter and enhancer. Treatment of the transfected cells with transition metallic ions (cadmium, cobalt, and zinc) or sodium arsenite produced increases in activities of luciferase or chloramphenicol acetyl transferase, relative to beta-galactosidase, and this activity mapped to the first 122 base pairs of the promoter. Although heme has recently been reported to induce the endogenous metallothionein gene in chick embryo liver cells, 10-50 microM heme did not increase reporter gene activities in transfected cells. Nevertheless, the heme-dependent induction of endogenous heme oxygenase-1 in these cells was normal. We conclude that the heme-dependent induction of the liver metallothionein gene depends upon DNA region(s) outside the regulatory region of the chick metallothionein gene studied here and that elements within the first 122 base pairs of the metallothionein promoter are sufficient to confer responsiveness to transition metals or sodium arsenite.
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PMID:Regulation of metallothionein gene expression. Studies in transfected primary cultures of chick embryo liver cells. 887 98

Because metallothionein (MT) is elevated and may be protective in UV-irradiated skin, we have studied the effects of UV and other agents on MT transcription using the sheep MT 1A promoter, linked to the beta-galactosidase gene and stably transfected into human cell lines. beta-galactosidase reporter activity was inducible by adding Zn2+ ions to the medium (100 microM for 2-4 h). Two differentiating agents, butyric acid and azelaic bishydroxamic acid (ABHA), significantly increased the response to Zn2+ in a melanoma cell line (MM96L-gal). UVB (280-315 nm) had two distinct, time-dependent effects. During the first 4 h after irradiation, high doses of UVB inhibited induction by Zn2+, an effect that was made more acute by simultaneous exposure to the differentiating agents. These changes in reporter activity were not due to alterations in Zn2+ transport into the cell. The UVB-depressed MT response subsequently recovered and by 24 h was double the control, yet remained sensitive to ABHA. Reporter activity in transfected HeLa cells differed from that in MM96L, being depressed 4 and 24 h after UVB and insensitive to ABHA at both times. Galactosidase reporter activity driven by non-MT promoters was not affected by these treatments. Dependence of MT transcriptional activity on UV-related DNA damage could be inferred because equitoxic UVC (254 nm) affected the response to Zn2+ in a similar fashion, whereas UVA, cisplatin and a methylating agent had no effect. The MT response was partly dependent on the PKC signal transduction pathway because it was inhibited by phorbol ester in HeLa, and by bisindolyl maleimide in HeLa and MM96L. The biphasic MT transcriptional response may model a signal transduction pathway that gives an early, depressed response to acute UV damage, with exacerbation by concurrent differentiation stimuli, but switches to a positive, cell-specific and potentially protective response at later times.
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PMID:Biphasic response of the metallothionein promoter to ultraviolet radiation in human melanoma cells. 907 40

The hydrolysis of o-nitrophenyl galactopyranoside and lactose by beta-D-galactosidase from Kluyveromyces lactis was enhanced by the addition of Mg2+ and Mn2+, but the rates of activation by each metal on both substrates were not the same. The Co2+, Zn2+, and Ni2+ activated the o-nitrophenyl galactopyranoside-hydrolyzing activity of the enzyme, but these same metals inhibited the lactose-hydrolyzing activity. The addition of Mg2+ and EDTA to the assay buffer increased the hydrolysis of o-nitrophenyl galactopyranoside and lactose at different rates. The responses of o-nitrophenyl galactopyranoside and lactose to the enzyme activity were different as a function of pH. The hydrolyzing activity toward both substrates also was influenced by the concentration of the phosphate in the assay buffer. However, the profile of the enzyme activity toward each substrate was different as a function of concentration. Because the assay of beta-galactosidase using o-nitrophenyl galactopyranoside is fast and convenient, the estimation of lactose-hydrolyzing activity of the enzyme has frequently been made based on the assay of o-nitrophenyl galactopyranoside hydrolysis. As shown in this study, a slight change in the conditions of the assay system and the enzyme application may cause changes in the ability of the enzyme to hydrolyze both lactose and o-nitrophenyl galactopyranoside. The change in o-nitrophenyl galactopyranoside-hydrolyzing activity is not always consistent with that of the lactose-hydrolyzing activity under the given condition, which may cause an inaccurate estimation of the enzyme activity in the enzyme preparation as well as in actual applications of the enzyme.
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PMID:Differences in the hydrolysis of lactose and other substrates by beta-D-galactosidase from Kluyveromyces lactis. 936 Nov 98

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.
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PMID:New functions for the three subunits of the CzcCBA cation-proton antiporter. 937 29


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