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)

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.
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PMID:Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria. 193 19

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase. 211 90

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.
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PMID:Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+. 211 47

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.
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PMID:Purification and properties of two lactose hydrolases from Trichosporon cutaneum. 212 10

The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.
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PMID:Thermal denaturation of beta-galactosidase and of two site-specific mutants. 212 99

The components of an HPLC system can be easily reassembled into an instrument for transient-state enzyme kinetic experiments based on the continuous-flow technique. The scheme of reassembly, operation and data evaluation is described in detail for the Pharmacia FPLC system. The working quality of the suggested scheme was tested using a well-studied process of bacterial beta-galactosidase activation by Mg2+ ions. The found parameters were in a good agreement with the literature data. The pulse-flow mode was developed for the delivery of working solutions to reduce the required quantities of enzyme and/or other reaction components. The work presents new approach to HPLC modules as building blocks for sophisticated liquid flow reactors, such as automatic solid phase synthesizers, sequencers and so on.
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PMID:Adaptation of an HPLC system for transient-state enzyme kinetic experiments: pulse-flow method. 212 9

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.
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PMID:Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100. 218 Sep 49

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage phi X174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of beta-galactosidase in the supernatant fraction of protein-E-lysed cells. Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein-E-mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.
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PMID:Biochemical characterization of phi X174-protein-E-mediated lysis of Escherichia coli. 252 90

A Mg2+-dependent ATPase activity has been purified from trout sperm axonemes which has properties characteristic of a dynein ATPase. A polyclonal antiserum prepared against the dynein heavy chains has been used to isolate dynein heavy chain (DYHC) cDNAs from a trout testis lambda gt11 cDNA expression library. beta-galactosidase fusion proteins produced in lambda gt11 by these trout cDNAs cross-reacted with a heterologous anti-sea urchin dynein antiserum. Northern blot analyses demonstrated that the RNA transcripts detected have sizes (7.5 - 12 kb) consistent with those expected for the dynein heavy chains. All the DYHC cDNAs encode portions of a highly unusual DNA coding sequence comprised of 21 bp direct repeats. The predicted open reading frame of this repeat is Ile/Leu-His-Val-Ile-Gln-Tyr-Ser and is characteristic of an extensive alpha-helical coiled-coil domain. The presence of an in-frame translation termination codon indicates that this domain is located at the carboxyl-terminus of the DYHC. Southern blot analyses demonstrated a low, if not single, copy number for this gene and conservation of this domain in other vertebrates. DYHC transcripts reach their highest level in testis, but are also abundant in brain tissue.
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PMID:Isolation of dynein heavy chain cDNAs from trout testis which predict an extensive carboxyl-terminal alpha-helical coiled-coil domain. 252 45

The activities of acid phosphatase, hexosaminidase, beta-galactosidase, Mg2+-stimulated Na+K+ATPase, fumarase and ATP:citrate lyase were measured in grey matter of rabbit spinal cord 7-8 days after intra-ventricular or intra-cisternal injection of aluminium. RNA, DNA, and water content were measured in whole spinal cords. Choline acetyltransferase (CAT) and acetylcholinesterase were assayed in dorsal grey matter of the cord, which contained no aluminium-induced neurofilament accumulations (NFAs), and ventral grey matter, which had large numbers of such NFAs. CAT was also assayed in the hypoglossal nerve. None of these measures were consistently altered in the aluminium treated rabbits, although the activity of beta-galactosidase was increased in the NFA-free caudate nucleus of rabbits given aluminium intra-ventricularly, possibly due to the presence of phagocytes on the ventricular surface of the caudate. It is concluded that neither aluminium nor its induced NFAs has a gross effect on neuronal metabolism within 7-8 days.
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PMID:Biochemical studies on rabbits with aluminium induced neurofilament accumulations. 298 21


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