scholarly journals Thermal denaturation of subtilisin-SSI complex analyzed by SDS-PAGE.

1990 ◽  
Vol 54 (2) ◽  
pp. 563-565
Author(s):  
Tsutomu ARAKAWA ◽  
Thomas P. HORAN
Keyword(s):  
Thermal Denaturation ◽  
Sds Page

scholarly journals Molecular mechanisms of the irreversible thermal denaturation of guinea-pig liver transglutaminase

1990 ◽  
Vol 266 (2) ◽  
pp. 487-490 ◽  
Author(s):  
S Nury ◽  
J C Meunier
Keyword(s):  
Thermal Denaturation ◽  
Molecular Mechanisms ◽  
Molecular Events ◽  
Sds Page ◽  
Irreversible Denaturation ◽  
Enzyme Molecules ◽  
Kinetics Of ◽  
Guinea Pig Liver ◽  
Step Mechanism ◽  
Rule Out

When transglutaminase is heated at temperatures above 40 degrees C, it loses its activity according to a two-step mechanism [Nury, Meunier & Mouranche (1989) Eur. J. Biochem. 180, 161-166]: N→X(TD)→D However, the nature of the molecular events responsible for the irreversible denaturation is still unknown. Investigation of the effects of dithiothreitol and 5,5′-dithiobis-2-nitrobenzoate on the kinetics of inactivation, titrations of ammonia released by deamidation and of thiol groups on the native and denatured enzymes and SDS/PAGE rule out the involvement of covalent processes during the denaturation of transglutaminase at 55 degrees C and pH 7. Of the two possible kinds of non-covalent events, i.e. unfolding of the polypeptide chain and aggregation of enzyme molecules, we show that both occur, though only the former process is responsible for the denaturation. The latter process, aggregation, follows the unfolding of the molecules.

scholarly journals Evidence of β-sheet structure induced kinetic stability of papain upon thermal and sodium dodecyl sulphate denaturation

2015 ◽  
Vol 80 (5) ◽  
pp. 613-625 ◽  
Author(s):  
Brankica Raskovic ◽  
Nikolina Babic ◽  
Jelena Korac ◽  
Natalija Polovic
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Thermal Denaturation ◽  
Thermal Inactivation ◽  
Elevated Temperatures ◽  
Sodium Dodecyl ◽  
High Activation ◽  
Rich Domain ◽  
Sds Page ◽  
Ft Ir ◽  
Β Sheet

Papain is a protease that consists of ?-helical and ?-sheet domains which unfold almost independently. Both, papain considerable thermal stability and sodium dodecyl sulphate (SDS) resistance have been shown. However, the ability of each domain to unfold upon thermal and SDS denaturation has never been studied. This work shows that fruit papain has slightly higher thermal inactivation resistance when it is compared to stem papain with rather high activation energy (Ea) of 223 ? 16 kJmol-1 and Tm50 value of 79 ? 2 ?C. SDS resistance of fruit papain was estimated by SDS-PAGE analysis and activity staining. It has been noted that, in the presence of SDS, unless heat energy was applied in order to unfold papain, the protein remained active. Furthermore, it has been proven via Fourier transform infrared spectroscopy (FT-IR) that ?-helical domain of fruit papain is more prone to unfolding at elevated temperatures and in the presence of SDS then ?-sheet rich domain. Thermal denaturation of papain without detergent present led to accelerated formation of aggregation specific intermolecular ?-sheets as compared to native protein. Presented results are both, of fundamental and application importance.

scholarly journals Isolation and Characterisation of Major and Minor Collagens from Hyaline Cartilage of Hoki (Macruronus novaezelandiae)

Marine Drugs ◽  
2019 ◽  
Vol 17 (4) ◽  
pp. 223 ◽  
Author(s):  
Cumming ◽  
Hall ◽  
Hofman
Keyword(s):  
Thermal Denaturation ◽  
Collagen Type ◽  
Hyaline Cartilage ◽  
Type Ii ◽  
Collagen Type Ii ◽  
Salt Precipitation ◽  
Nasal Cartilage ◽  
Sds Page ◽  
Native Collagen ◽  
First Time

The composition and properties of collagen in teleost (bony fish) cartilage have never been studied. In this study, we aimed to identify and characterise all collagen species in the nasal cartilage of hoki (Macruronus novaezelandiae). Four native collagen species were extracted using two techniques, and isolated with differential salt precipitation. We were able to assign the identity of three of these collagen species on the basis of solubility, SDS-PAGE and amino acid analyses. We found that hoki cartilage contains the major collagen, type II, and the minor collagens, type IX and type XI, which are homologous to those found in mammal and chicken cartilage. Using these extraction protocols, we also isolated a full-length type IX collagen from cartilage for the first time. In addition, we detected a 90 kDa, highly glycosylated collagen that has not been identified in any other species. For each isolate, structural and biochemical characterisations were performed using circular dichroism and Fourier transform infrared spectroscopy analyses, and the thermal denaturation properties were determined. Our results showed that the properties of hoki cartilage-derived collagens are similar to those of collagens in mammalian cartilage, indicating that teleost cartilage could provide biological ingredients for the development of biomaterials to treat cartilage-related illnesses.

scholarly journals Thermal Denaturation of Subtilisin-SSI Complex Analyzed by SDS-PAGE

1990 ◽  
Vol 54 (2) ◽  
pp. 563-565
Author(s):  
Tsutomu Arakawa ◽  
Thomas P. Horan
Keyword(s):  
Thermal Denaturation ◽  
Sds Page

scholarly journals Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides

1993 ◽  
Vol 295 (3) ◽  
pp. 645-648 ◽  
Author(s):  
M M Altamirano ◽  
J A Plumbridge ◽  
H A Barba ◽  
M L Calcagno
Keyword(s):  
Thermal Denaturation ◽  
Cysteine Residues ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Urea Denaturation ◽  
Disulphide Bonds ◽  
Oligomeric Protein ◽  
Sds Page ◽  
Mutant Forms ◽  
Allosteric Properties

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

1988 ◽  
Vol 46 ◽  
pp. 30-31
Author(s):  
E. T. O'Toole ◽  
R. R. Hantgan ◽  
J. C. Lewis
Keyword(s):  
Whole Blood ◽  
Colloidal Gold ◽  
Blood Platelets ◽  
Cell Contact ◽  
Computer Assisted ◽  
Adherent Cells ◽  
Differential Centrifugation ◽  
Computer Reconstruction ◽  
Sds Page ◽  
Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

1999 ◽  
Vol 82 (11) ◽  
pp. 1428-1432 ◽  
Author(s):  
Cheryl Scott ◽  
Francesco Salerno ◽  
Elettra Lorenzano ◽  
Werner Müller-Esterl ◽  
Angelo Agostoni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Liver Disease ◽  
Liver Function ◽  
Plasma Levels ◽  
Plasma Concentrations ◽  
Normal Subjects ◽  
Low Molecular Weight ◽  
Major Band ◽  
Sds Page ◽  
Normal Controls

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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