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)

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56

The ultrastructure of choline acetyltransferase (ChAT)-immunoreactive neurons in the laterodorsal tegmental nucleus (TLD) of the rat was investigated by immunohistochemical techniques. The immunoreactive neurons were medium to large in size, with a few elongated dendrites, contained well-developed cytoplasm, and a nucleus with deep infoldings. They received many nonimmunoreactive, mostly asymmetric synaptic inputs on their soma and dendrites. ChAT-immunoreactive, usually myelinated, axons were occasionally seen in TLD. Only one immunoreactive axon terminal was observed within TLD, and it made synaptic contact with a nonimmunoreactive neuronal perikaryon. The synaptic interactions between ChAT-immunoreactive neurons and tyrosine hydroxylase (TH)-immunoreactive fibers in the TLD were investigated with a double immunohistochemical staining method. ChAT-immunoreactivity detected with a beta-galactosidase method was light blue-green in the light microscope and formed dot-like electron dense particles at the electron microscopic level. TH-immunoreactivity, visualized with a nickel-enhanced immunoperoxidase method, was dark blue-black in the light microscope and diffusely opaque in the electron microscope. Therefore, the difference between these two kinds of immunoreactivity could be quite easily distinguished at both light and electron microscopic levels. In the light microscope, TH-positive fibers were often closely apposed to ChAT-immunoreactive cell bodies and dendrites in TLD. In the electron microscope, the cell soma and proximal dendrites of ChAT-immunoreactive neurons received synaptic contacts from TH-immunoreactive axon terminals. These results provide a morphological basis for catecholaminergic regulation of the cholinergic reticular system.
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PMID:Ultrastructure of cholinergic neurons in the laterodorsal tegmental nucleus of the rat: interaction with catecholamine fibers. 139 18

Transcription of the corynebacteriophage diphtheria tox operon has been shown to be regulated through a corynebacterial determined factor DtxR. The dtxR gene has been recently cloned and expressed in Escherichia coli, and shown to regulate the expression of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion. Tao et al. (Tao, X., Boyd, J., and Murphy, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5897-5901) have recently shown by gel mobility shift assay that the binding of DtxR to the tox operator requires a divalent heavy metal ion, as well as the 27-base pair interrupted palindromic sequence. We now show the activation of DtxR in the presence of Co2+, Fe2+, or Mn2+ results in the protection of a 33- and 27-nucleotide region on the coding and non-coding strand from DNase I digestion, respectively. DtxR is also activated in the presence of Ni2+; however, this metalloregulatory factor is only weakly activated by Zn2+. The diphtheria tox regulatory region protected from DNase I digestion in the presence of activated DtxR encompasses the 27-base pair interrupted palindromic sequence.
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PMID:Binding of the metalloregulatory protein DtxR to the diphtheria tox operator requires a divalent heavy metal ion and protects the palindromic sequence from DNase I digestion. 140 Apr 85

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).
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PMID:CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. 145 58

Salmonella typhimurium contains three distinct transport systems (CorA, MgtA, and MgtB) that move Mg2+ across the cytoplasmic membrane. Mutant strains containing only one of these three systems have been constructed and used to study each system in isolation. Characterization of these systems has been hampered, however, by the need to use 28Mg2+, a relatively unavailable, extremely expensive, and short lived radioisotope. This paper reports that 63Ni2+ is transported into the cell by all three of the S typhimurium Mg2+ transport systems. In a strain deficient in all three systems, uptake of 63Ni2+ was undetectable under the conditions used. Comparison of 63Ni2+ uptake kinetics and inhibition of 63Ni2+ transport by other divalent cations suggest that Ni2+ can be used as an analog of Mg2+ in the study of these three transport systems. Using 63Ni2+ to measure uptake, the effect of Mg2+ levels in the growth medium on transport by each system was tested. Transport by the CorA system was unaffected by changes in the amount of Mg2+ in the growth medium. In contrast, uptake via MgtA and MgtB was significantly increased in cells grown in 10 microM extracellular Mg2+ compared to cells grown in 10 mM Mg2+. The increases in uptake were the result of increases in Vmax without change in Km. This result suggests that, in low Mg2+ medium, cells contained higher levels of the transporters. Production of beta-galactosidase from mgtA::lacZ and mgtB::lacZ but not corA::lacZ fusions was also increased when cells were grown in low extracellular concentrations of Mg2+ indicating that the regulation occurs at the level of transcription. Expression of beta-galactosidase was also inhibited by the addition of other divalent cations including Ca2+ and Mn2+. Regulation of transcription from the mgtA and mgtB promoters was similar over the range of extracellular Mg2+ concentrations from 10 microM to 10 mM. At 1 microM, however, transcription from the mgtB promoter, as measured by beta-galactosidase levels in a mgtB::lacZ transcriptional fusion strain, was increased over 800-fold, and Ca2+ could no longer inhibit transcription effectively. In contrast, growth at 1 microM extracellular Mg2+ increased transcription from the mgtA promoter only about 30-fold and Ca2+ could still inhibit this increase. These results suggest that at least two distinct mechanisms are responsible for regulation of the mgtA and mgtB transcription in response to extracellular cation concentration.
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PMID:Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression. 189 38

The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1. A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for beta-galactosidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A. eutrophus gene hoxN. An E. coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction. A broad-host-range derivative of pCH307 was shown to complement an A. eutrophus HoxN- mutant.
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PMID:Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus. 203 63

The phototoxicities of six metalloporphyrin dimethylesters (i.e. cobalt (Co), copper (Cu), manganese (Mn), nickel (Ni), tin (Sn) and zinc (Zn) were investigated. Hemolysis of human erythrocytes and inactivation of two enzymes (acetylcholinesterase and beta-galactosidase) were used to assess the phototoxic efficacy of these metal chelates. Tin protoporphyrin (SnPP), the only porphyrin found to hemolyze erythrocytes at a concentration of 40 microM (radiation dose, 230 kJ m-2), was much less efficient than either free protoporphyrin IX or hematoporphyrin. SnPP completely inactivated beta-galactosidase at concentrations above 15 microM (radiation dose, 75 kJ m-2) and drastically interfered with acetylcholinesterase activity at a concentration of 150 microM (radiation dose, 75 kJ m-2). CoPP, CuPP, MnPP, NiPP and ZnPP were ineffective photohemolytic agents at 40 microM (radiation dose, 230 kJ m-2), but inactivated acetylcholinesterase and beta-galactosidase activity to varying degrees. These results suggest that (i) metal ions reduce the phototoxicity of protoporphyrin IX, (ii) different metal ions reduce the phototoxic activity of protoporphyrin IX to different degrees and (iii) the biological activities of the various metal complexes vary in different assay systems.
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PMID:Metalloporphyrin phototoxicity. 212 21

The Mu dl (ApR lac) bacteriophage was used to generate mutants of Escherichia coli which were defective in formate hydrogenlyase. Three mutants were chosen for further analysis: they lacked hydrogenase (hydrogen: benzyl viologen oxidoreductase) activity, but produced normal levels of fumarate reductase activity and two- to three-fold reduced levels of benzyl viologen (BV)-dependent formate dehydrogenase activity. Two of them (hydC) were shown to contain about 4-fold reduced amounts of formate hydrogenlyase and fumarate-dependent H2 uptake activities. The third one (hydD) was totally devoid of both activities. Their insertion sites were located at 77 min on the E. coli map. Subdivision of these mutants into two classes was subsequently based on the restoration capacity of hydrogenase activity with high concentration of nickel in the growth media. Addition of 500 microM NiCl2 led to a complete recovery of hydrogenase activity, and to the concomitant restoration of normal BV-linked formate dehydrogenase, formate hydrogenlyase and fumarate-dependent H2 uptake activities in the hydC mutants. The hydD mutant was insensitive to the effect of nickel. Expression of the lac operon in hydC and hydD mutants was induced by anaerobiosis. It was not increased by the addition of formate under anaerobic conditions. The presence of nitrate resulted in slightly reduced beta-galactosidase activities in the hydC mutants, whereas those found in the hydD mutant reached only one third of the level obtained in its absence. Fumarate had no effect on both classes. Moreover, in contrast to the hydD locus, the hydC::Mu dl fusions were found to be dependent upon the positive control exerted by the nirR gene product and were totally repressed by an excess of nickel. In addition, the low levels of overall hydrogenase-dependent activities found in a nirR strain were also relieved by the presence of nickel. Our results strongly suggest that the pleiotropic regulatory gene nirR is essential for the expression of a gene (hydC) involved in either transport or processing of nickel in the cell, whose alteration leads to a loss of hydrogenase activity.
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PMID:Genetic and physiological characterization of new Escherichia coli mutants impaired in hydrogenase activity. 308 8

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).
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PMID:Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency. 680 Mar 55

beta-galactosidase (Escherichia coli) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+. Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type beta-galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type beta-galactosidase activity, the substituted enzyme has very different divalent metal interactions. A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme. The inactivation of E461H-beta-galactosidase caused by Mg2+ took about 20 min. Reactivation by removal of the divalent metal took about 60 min. Interaction with Mg2+ was about 10(7)-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values. "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-beta-galactosidase at pH values above 6 with both o-nitrophenyl beta-D-galactopyranoside and p-nitrophenyl beta-D-galactopyranoside in both the presence and absence of Mg2+. Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-beta-galactosidase (apparent pKa was about 6.8).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation. 757 31


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