scholarly journals Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

scholarly journals The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

1976 ◽  
Vol 155 (2) ◽  
pp. 325-330 ◽  
Author(s):  
B Robson ◽  
R H. Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Tyrosine Residues ◽  
Low Concentrations ◽  
Unfolded State ◽  
Solvent Molecules ◽  
Homogeneous Preparation

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

scholarly journals Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 641-646 ◽  
Author(s):  
David Snary ◽  
Adrian Allen ◽  
Roger H. Pain
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Sedimentation Coefficient ◽  
Water Structure ◽  
Water Soluble ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Gastric Mucus ◽  
Caesium Chloride ◽  
Low Concentrations

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S→33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs+and Cl−ions on water structure and the water–mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s025,w (33S→26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.

scholarly journals Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site

1995 ◽  
Vol 305 (2) ◽  
pp. 379-384 ◽  
Author(s):  
H J Yang ◽  
C L Tsou
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Ribonuclease A ◽  
Proteinase K ◽  
Peptide Fragments ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Low Concentrations ◽  
Partial Unfolding ◽  
Absorption Measurements

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Active-Site Heterogeneity of Lactate Dehydrogenase

ACS Catalysis ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 4236-4246 ◽  
Author(s):  
He Yin ◽  
Hui Li ◽  
Adam Grofe ◽  
Jiali Gao
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Site Heterogeneity

scholarly journals Effects of a Polyoxyethylene Detergent on Platelet Function

1977 ◽  
Author(s):  
P.G. Barton
Keyword(s):  
Lactate Dehydrogenase ◽  
Platelet Rich Plasma ◽  
Secondary Phase ◽  
Human Platelets ◽  
Brij 58 ◽  
Low Concentrations ◽  
High Concentrations ◽  
Glass Surfaces ◽  
Induced Aggregation ◽  
Membrane Destabilization

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet rich plasma but had no effect on primary aggregation.Thrombin-induced aggregation of washed human platelets suspended in Tyrode’s buffer was inhibited after incubation of cells with 4.5 × 10-6M detergent. Development of prothrombin-converting activity and efflux of [14C]-serotonin, 45Ca2+ ions and labile endoperoxides were abolished concomitantly. Aggregation of washed platelets by collagen or sodium arachidonate and the attachment of cells to clean glass surfaces were also inhibited by the same concentration of Brij 58 that inhibited thrombin aggregation. This concentration of Brij 58 did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 10-4 M, lysed the cells liberating all of their serotonin, Ca2+ and lactate dehydrogenase. These results suggest that low concentrations of Brij 58 stabilize a membrane conformation against the action of platelet stimulatory agents while high concentrations produce membrane destabilization and cell lysis. The presence of albumin (BSA) in the suspending fluid increased by tenfold the concentrations of detergent required to “elicit these effects and this could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]-acetylated Brij 58.

Probing the Active Site of a Diels−Alderase Ribozyme by Photoaffinity Cross-Linking

2008 ◽  
Vol 130 (27) ◽  
pp. 8594-8595 ◽  
Author(s):  
Richard Wombacher ◽  
Andres Jäschke
Keyword(s):  
Active Site ◽  
Cross Linking

An intersubunit interaction at the active site of D-ribulose-1,5-bisphosphate carboxylase/oxygenase as revealed by cross-linking and site-directed mutagenesis

Biochemistry ◽  
1987 ◽  
Vol 26 (15) ◽  
pp. 4599-4604 ◽  
Author(s):  
Eva H. Lee ◽  
Thomas S. Soper ◽  
Richard J. Mural ◽  
Claude D. Stringer ◽  
Fred C. Hartman
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Bisphosphate Carboxylase
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