scholarly journals Catalytic activity of bovine glutamate dehydrogenase requires a hexamer structure

1984 ◽  
Vol 217 (1) ◽  
pp. 327-330 ◽  
Author(s):  
E T Bell ◽  
J E Bell
Keyword(s):  
Catalytic Activity ◽  
Glutamate Dehydrogenase ◽  
Guanidinium Chloride ◽  
Full Activity ◽  
Chloride Concentrations

Previous workers have shown that the hexamers of glutamate dehydrogenase are dissociated first into trimers and subsequently into monomers by increasing guanidinium chloride concentrations. In renaturation experiments it is shown that trimers of glutamate dehydrogenase can be reassociated to give the hexamer form of the enzyme, with full regain of activity. Monomeric subunits produced at high guanidinium chloride concentrations cannot be renatured. The trimer form of the enzyme is shown to have no catalytic activity, although the hexamer form in guanidinium chloride has full activity.

Comparisons of 17 lots of 2-oxoglutarate, and specifications for use of this substrate in reference methods.

1985 ◽  
Vol 31 (6) ◽  
pp. 812-818
Author(s):  
P N Bowers ◽  
G N Bowers ◽  
R B McComb
Keyword(s):  
Catalytic Activity ◽  
Liquid Chromatography ◽  
Glutamate Dehydrogenase ◽  
Spectral Characteristics ◽  
Poor Quality ◽  
Activity Concentrations ◽  
Reference Methods ◽  
Specific Analysis ◽  
Quality Material ◽  
Salt Forms

Abstract We examined 17 lots of 2-oxoglutarate (seven acid forms, three K salt forms, and seven Na salt forms), obtained from eight commercial suppliers, for suitability for measuring aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in human serum. Measurements of the catalytic activity concentrations of these two aminotransferases with each of these 17 preparations were not sufficiently sensitive to distinguish good from poor-quality material. Thus, we ranked these lots for purity, by specific analysis with glutamate dehydrogenase and by liquid chromatography, and determined the water content, acid content, and spectral characteristics of each. On the basis of a 2-oxoglutarate assay value by glutamate dehydrogenase of 98% or greater, we considered seven of the preparations acceptable and 10 unacceptable. The molar absorptivities (L X mol-1 X cm-1, mean +/- SD) of the seven acceptable lots in 1 mol/L HCl were: epsilon 325 nm = 9.12 +/- 0.02 (CV = 0.2%), epsilon 279 nm = 2.63 +/- 0.23 (CV = 9.9%), and epsilon 245 nm = 37.9 +/- 4.1 (CV = 10.9%). Use of these spectrophotometric limits alone unambiguously distinguished the inferior lots of 2-oxoglutarate. We urge the inclusion of detailed spectrophotometric specifications for 2-oxoglutarate in Reference Methods for aminotransferase measurements.

scholarly journals Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

2021 ◽  
Author(s):  
Luning Yu ◽  
Xinqi Kang ◽  
Mohammad Amin Alibakhshi ◽  
Mikhail Pavlenok ◽  
Michael Niederweis ◽  
...  
Keyword(s):  
Guanidinium Chloride ◽  
Polymer Bilayers ◽  
Protein Channel ◽  
Chloride Concentrations

scholarly journals Identification by n.m.r. spectroscopy of a stable intermediate structure in the unfolding of staphylococcal β-lactamase

1983 ◽  
Vol 215 (3) ◽  
pp. 525-529 ◽  
Author(s):  
R M Thomas ◽  
J Feeney ◽  
R B Nicholson ◽  
R H Pain ◽  
G C K Roberts
Keyword(s):  
Intermediate Structure ◽  
Beta Lactamase ◽  
Flow Properties ◽  
Guanidinium Chloride ◽  
Detailed Characterization ◽  
Local Sequence ◽  
Sequence Effects ◽  
Proton Resonances ◽  
Chloride Concentrations

The unfolding of beta-lactamase (penicillinase) from Staphylococcus aureus by guanidinium chloride was followed by using n.m.r. spectroscopy. On the basis of the observation of resonances corresponding to histidine, tyrosine and other amino acid side chains, the existence of a stable partially folded species was demonstrated. These experiments provide detailed characterization of the intermediate that confirms and extends previous characterization by absorption and c.d. spectroscopy and by flow properties. In addition, they show that residues in the N-terminal third of the molecule are affected by the native-to-intermediate transition. Persistent non-equivalence of the two imidazole C2 proton resonances at high guanidinium chloride concentrations is discussed in terms of local sequence effects on the chemical shift.

scholarly journals The denaturation of rabbit muscle phosphorylase b by guanidinium chloride

1983 ◽  
Vol 213 (3) ◽  
pp. 595-602 ◽  
Author(s):  
N C Price ◽  
E Stevens
Keyword(s):  
Absorption Spectroscopy ◽  
Structural Changes ◽  
Thiol Group ◽  
Rabbit Muscle ◽  
Guanidinium Chloride ◽  
Protein Fluorescence ◽  
Initial Concentration ◽  
Full Activity ◽  
Folded Structure ◽  
Muscle Phosphorylase

The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.

Intersubunit Interaction Induced by Subunit Rearrangement Is Essential for the Catalytic Activity of the Hyperthermophilic Glutamate Dehydrogenase fromPyrobaculum islandicum†

Biochemistry ◽  
2005 ◽  
Vol 44 (46) ◽  
pp. 15304-15313 ◽  
Author(s):  
Shuichiro Goda ◽  
Masaki Kojima ◽  
Yoshimi Nishikawa ◽  
Chizu Kujo ◽  
Ryushi Kawakami ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Glutamate Dehydrogenase

scholarly journals The effect of N-bromosuccinimide on the sub-unit structure of avidin and its complexes with biotin

1968 ◽  
Vol 110 (1) ◽  
pp. 59-66 ◽  
Author(s):  
N. M. Green ◽  
M. E. Ross
Keyword(s):  
Binding Sites ◽  
Oxidation Products ◽  
Guanidinium Chloride ◽  
Unit Structure ◽  
Tryptophan Residues ◽  
Biotin Binding ◽  
Tetrameric Complex ◽  
Chloride Concentrations ◽  
Avidin Biotin Complex

1. Each molecule of biotin bound to avidin protected four tryptophan residues from oxidation by N-bromosuccinimide, regardless of the occupancy of neighbouring binding sites in the four-sub-unit avidin molecule. 2. The oxidation products from avidin molecules in which some of the sites were occupied were separated on columns of Sephadex G-100. In the absence of biotin, oxidized avidin broke down into sub-units, which partly aggregated. When some of the sites were occupied by biotin, the only detectable products were completely oxidized avidin (sub-units and large aggregates) and unoxidized avidin–biotin complex (tetramer). Since the biotin-containing sub-units were randomly distributed before oxidation took place, they must have dissociated from the molecules containing oxidized sub-units and then reassociated to form the tetrameric avidin–biotin complex. 3. This reassociation still occurred in 3·5m-guanidinium chloride, which prevents the reassociation of unoccupied sub-units. During their brief existence in this medium, the sub-units of avidin–biotin complex were protected from oxidation by N-bromo-succinimide to the same extent as was the tetrameric complex. 4. It is concluded that sub-units of avidin–biotin complex do not readily lose their biotin, even in 3·5m-guanidinium chloride, and that monomeric biotin–binding species are probably present in solutions of avidin sub-units at guanidinium chloride concentrations between 3·0m and 3·5m.

scholarly journals Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers

1978 ◽  
Vol 175 (3) ◽  
pp. 1125-1133 ◽  
Author(s):  
D H Watson ◽  
J C Wootton
Keyword(s):  
Catalytic Activity ◽  
Affinity Chromatography ◽  
Neurospora Crassa ◽  
Glutamate Dehydrogenase ◽  
Binomial Distribution ◽  
Hybridization Method ◽  
Apparent Absence ◽  
Freeze Thaw ◽  
Monomer Ratio ◽  
Conformational Constraints

The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.

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