Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: Evidence for two distinct enzyme forms

2001 ◽  
Vol 79 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Bradley J Thatcher ◽  
Kenneth B Storey
Keyword(s):  
Body Temperature ◽  
Glutamate Dehydrogenase ◽  
Ground Squirrels ◽  
Size Exclusion ◽  
Kinetic Properties ◽  
Molecular Weights ◽  
Spermophilus Richardsonii ◽  
Subunit Molecular Weight ◽  
A Subunit ◽  
Size Exclusion Hplc

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37°C body temperature) and hibernating (torpid, 5°C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5 ± 2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335 ± 5 kDa for euthermic and 320 ± 5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and α-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5°C). For example, the Km for α-ketoglutarate of euthermic GDH was higher at 5°C (3.66 ± 0.34 mM) than at 37°C (0.10 ± 0.01 mM), whereas hibernator GDH had a higher affinity for α-ketoglutarate at 5°C (Km was 0.98 ± 0.08 mM at 37°C and 0.43 ± 0.02 mM at 5°C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37°C and weakest with hibernator GDH at 5°C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.Key words: mammalian hibernation, amino acid metabolism, temperature-dependent enzyme kinetics, Spermophilus richardsonii.

scholarly journals Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach

1983 ◽  
Vol 210 (1) ◽  
pp. 37-47 ◽  
Author(s):  
B T Pan ◽  
R Blostein ◽  
R M Johnstone
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Transferrin Receptor ◽  
Plasma Membranes ◽  
Plasma Membrane Proteins ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Cell Plasma ◽  
A Subunit

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.

scholarly journals Antibodies to the glutamate dehydrogenase ofPlasmodium falciparum

Parasitology ◽  
1986 ◽  
Vol 92 (2) ◽  
pp. 313-324 ◽  
Author(s):  
I. T. Ling ◽  
S. Cooksley ◽  
P. A. Bates ◽  
E. Hempelmann ◽  
R. J. M. Wilson
Keyword(s):  
Glutamate Dehydrogenase ◽  
Plasmodium Knowlesi ◽  
Bovine Liver ◽  
Sucrose Density Gradient ◽  
Subunit Molecular Weight ◽  
Bacterial Enzyme ◽  
Ofplasmodium Falciparum ◽  
A Subunit ◽  
Polyclonal Antisera ◽  
Hexameric Form

SUMMARYPolyclonal antisera raised againstPlasmodium knowlesireacted with (1) NADP-specific glutamate dehydrogenase (GLDH) ofP. knowlesi, (2) GLDH ofP. falciparumand (3) GLDH ofProteus spp. The antisera did not react with NAD(P) GLDH from bovine liver. Polyclonal antisera raised against the GLDH ofProteus spp. cross-reacted with GLDH fromP. falciparum. Monoclonal antibodies (McAbs) obtained from mice immunized with Proteus GLDH were either specific for the bacterial enzyme or cross-reacted withP. falciparumGLDH. The selected McAbs did not react with GLDI-1 fromP. knowlesi,P. chabaudiorP. berghei. The GLDH ofP. falciparumwas shown to be a cytosolic protein (by FAT) with a subunit molecular weight of approximately 49000 Da (by immunoprecipitation) having a pre dominantly hexameric form (by sucrose density gradient). Implications of the conserved sequences of GLDHs and other enzymes are discussed.

A size-exclusion HPLC method for the determination of sodium chondroitin sulfate in pharmaceutical preparations

2003 ◽  
Vol 31 (6) ◽  
pp. 1229-1236 ◽  
Author(s):  
Don Woong Choi ◽  
Mi Jung Kim ◽  
Hee Sung Kim ◽  
Soo Hyun Chang ◽  
Gi Sook Jung ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Pharmaceutical Preparations ◽  
Hplc Method ◽  
Size Exclusion ◽  
Size Exclusion Hplc

scholarly journals Speciation of Water-Soluble Boron Compounds in Radish Roots by Size Exclusion HPLC/ICP-MS.

1996 ◽  
Vol 12 (4) ◽  
pp. 673-675 ◽  
Author(s):  
Toshiro MATSUNAGA ◽  
Tadashi ISHII ◽  
Hisao WATANABE
Keyword(s):  
Water Soluble ◽  
Size Exclusion ◽  
Boron Compounds ◽  
Icp Ms ◽  
Size Exclusion Hplc

Effects of central injection of biogenic amines during arousal from hibernation

1979 ◽  
Vol 236 (3) ◽  
pp. R162-R167 ◽  
Author(s):  
J. D. Glass ◽  
L. C. Wang
Keyword(s):  
Ambient Temperature ◽  
Specific Heat ◽  
Biogenic Amines ◽  
Heat Production ◽  
Ground Squirrels ◽  
Spermophilus Richardsonii ◽  
Simultaneous Measurements ◽  
Apparent Specific Heat ◽  
Intracerebroventricular Injections ◽  
Made In

Simultaneous measurements of heat production (HP) and heat loss (HL) and brain and rectal temperatures were made in Richardon's ground squirrels (Spermophilus richardsonii) rewarming from hibernation at an ambient temperature of 6.0 +/- 0.5 degrees C. Calculations from HP and HL measurements from control animals showed that due to differential rewarming, there was a reduction of apparent specific heat of the animal to 0.59 cal/g. degrees C. This resulted in an energy saving of 30%. Three intracerebroventricular injections of 5-hydroxytryptamine (5-HT) of 56 microgram each at brain temperatures of 10, 20, and 30 degrees C caused initial suppression of HP and a greater overall HL, which resulted in a slower rate of arousal as compared to the controls. Injections of norepinephrine (NE) of 12.5 microgram each at similar brain temperatures caused a greater rate of HP, which resulted in a faster rate of arousal as compared to the controls. The respective actions of 5-HT and NE on thermoregulation during rewarming are similar to those in some euthermic hibernators and nonhibernating species. Our data indicated that these substances evoke thermoregulatory responses during arousal in much the same manner as during normothermia.

scholarly journals Purification and characterization of cystatin from duck egg white.

1995 ◽  
Vol 42 (3) ◽  
pp. 351-356 ◽  
Author(s):  
M Warwas ◽  
J Gburek ◽  
J Osada ◽  
K Gołab
Keyword(s):  
Egg White ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Molecular Forms ◽  
Sds Page ◽  
Peptidase Inhibitor ◽  
Size Exclusion Hplc ◽  
Duck Egg ◽  
Chicken Cystatin

It is the second peptidase inhibitor, after ovostatin, which showing the same antipapain activity in egg white in different avian species implies differences in amino-acid sequences. Cystatin from duck egg white was purified by carboxymethylpapain affinity chromatography and size-exclusion HPLC. The purified inhibitor which showed partial identity in the immunodiffusion test with chicken egg white cystatin, had an apparent molecular mass of 9.3 kDa as determined by SDS/PAGE. IEF analysis revealed five molecular forms of pI in the range 7.8-8.4. The obtained cystatin was neither glycosylated nor phosphorylated as it is in the case of chicken cystatin. The determined Ki (0.005 +/- 0.001 nM) was similar to that reported for human and chicken cystatin C.

Synthesis and characterization of new, liquid, branched poly(methylvinylborosiloxanes)

e-Polymers ◽  
2013 ◽  
Vol 13 (1) ◽  
Author(s):  
Jerzy Chruściel ◽  
Marzena Fejdyś ◽  
Witold Fortuniak
Keyword(s):  
Functional Groups ◽  
Atomic Spectroscopy ◽  
Size Exclusion ◽  
Random Structure ◽  
Molecular Weights ◽  
Chemical Structures ◽  
Siloxane Oligomers ◽  
Exclusion Chromatography ◽  
Ether Solution

Abstract New liquid branched poly(methylvinylborosiloxanes) (br-PMVBS) of random structure were synthesized in three steps. By reacting boric acid with an excess of dimethyldichlorosilane (Me2SiCl2) in dry ether a “borosiloxane precursor”: tris(chlorodimethylsilyl) borate B(OSiMe2Cl)3 was prepared. In the second step of synthesis ether solution of B(OSiMe2Cl)3 was added to a mixture of appropriate organic chlorosilanes (Me2SiCl2, MeViSiCl2, MeSiCl3, and Me3SiCl) and all reagents were reacted with stoichiometric amounts of water, in the presence of pyridine (as an acceptor of HCl), in dry ether, at low temperature (usually at -10 to 0 C). In order to fully react (“to block”) trace silanol groups, reactions of intermediate PMVBS with additional batches of Me3SiCl were carried out in the third step, C5H5N·HCl was filtered off and washed with a dry ether. The solvent was distilled off from filtrates and low molecular weight siloxane oligomers were removed by a vacuum distillation at 130-150 C. Chemical structures of br-PMVBS were confirmed by elemental analysis and spectroscopic methods (FTIR, emission atomic spectroscopy ICP-AES, and NMR: 1H, 29Si and 11B). On the basis of analysis of their 29Si-NMR spectra the microstructure of polysiloxane chains was proposed. The prepared br-PMVBS had in their structures: triple branching borosiloxane units: BO1.5 and in some cases methylsiloxane moiety CH3SiO1.5 (T). They contained linkages: Si-O-Si, Si-O-B, vinyl(methyl)siloxane functional groups (CH2=CH)MeSiO (Dvi), dimethylsiloxane mers (CH3)2SiO (D), and non-reactive trimethylsiloxy terminal groups (CH3)3SiO0.5 (M), but they did not have: hydroxyl functional groups: Si-OH and B-OH, and sensitive to water B-O-B linkages. Molecular weights of br-PMVBS (Mn = 1500-3300 g/mol; Mw = 3800-7400 g/mol) and their polydispersity (Mw/Mn = 2.0-2.5) were determined by a size exclusion chromatography (SEC).

A rapid purification procedure for pyruvate kinase from the hyphal fungus Aspergillus nidulans

1988 ◽  
Vol 34 (10) ◽  
pp. 1154-1158 ◽  
Author(s):  
Harry C. M. Kester ◽  
Jos H. A. A. Uitzetter ◽  
Leo H. de Graaff ◽  
Jaap Visser
Keyword(s):  
Molecular Weight ◽  
Liquid Chromatography ◽  
Aspergillus Nidulans ◽  
Pyruvate Kinase ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Highly Active ◽  
A Subunit ◽  
Rapid Purification ◽  
Dye Affinity Chromatography

Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45–55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65 000 and an isoelectric point of 4.7, was used to determine the amino acid composition.

Sign in / Sign up

Export Citation Format

Share Document