Denaturation-Renaturation of the Fibrin-Stabilizing Factor XIII a-Chain Isolated from Human Placenta. Properties of the Native and Reconstituted Protein

Rinas, Ursula; Risse, Bernhard; Jaenicke, Rainer; Abel, Karl-Josef; Zettlmeissl, Gerd

doi:10.1515/bchm3.1990.371.1.49

Cited by 25 publications

(20 citation statements)

References 14 publications

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“…Since the conditions for protein renaturation vary between different proteins, we adapted the conditions that have been established for a homologous protein, plasma transglutaminase (i.e. coagulation factor XIII A chains) (25). The results demonstrated that 90 -95% of the protein remained soluble after renaturation.…”

Section: Resultsmentioning

confidence: 99%

“…Denaturation and Renaturation of GST-tTG and GST-⌬P345-The protocol for denaturation and renaturation was adapted from the procedures established for plasma transglutaminase (25). GST-tTG and GST-⌬P345 (100 g/ml) were denatured in a solution containing 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol/EDTA, and 6 M guanidinium HCl at room temperature for 1 h. Renaturation was performed by dialyzing the denatured GST-tTG and GST-⌬P345 either in a solution containing 20 mM Tris-Cl, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol/EDTA (buffer A) at 4°C for 16 h or in buffer A containing 1 M urea for 8 h and then in buffer A for additional 8 h. After dialysis, the samples were centrifuged at top speed in Eppendorf's microcentrifuge (model 5415C) for 30 min to remove the insoluble proteins.…”

Section: Quantitation Of Atp Hydrolysis and Analysis Of Reaction Prodmentioning

confidence: 99%

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C-terminal Deletion of Human Tissue Transglutaminase Enhances Magnesium-dependent GTP/ATPase Activity

Lai

Slaughter

Koropchak

et al. 1996

Journal of Biological Chemistry

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Tissue transglutaminase (tTG) exhibits a magnesiumdependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (⌬S538, ⌬E447, ⌬P345, ⌬C290, ⌬V228, and ⌬F185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent K m value for either GTP or ATP. In contrast, the K cat value for GTP was increased by 4.6-and 3-fold for the ⌬S538 and ⌬E447 mutants, respectively. The ⌬P345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1.9-fold for the ⌬C290, ⌬V228, and ⌬F185 mutants, respectively. The K cat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the ⌬P345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the ⌬S538 and ⌬E447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.Tissue transglutaminase (tTG) 1 is a unique member of the transglutaminase gene family in that it exhibits two distinct enzyme activities (1-3). The calcium-dependent transglutaminase activity (TGase) catalyzes the covalent modification of proteins by the formation of ␥-glutamyl-⑀-lysine bonds between proteins or polyamines (1, 2). The TGase activity is considered to be an important intracellular and extracellular reaction during apoptosis (4, 5), bone ossification (6), tissue repair (7), and tumor growth (8). TGase activity requires a calcium binding site and active site cysteine to form a thioester bond with the glutamine substrate (1, 2). The active site of human tTG is located at Cys-277, and the putative calcium binding site is located between amino acids 446 and 453 based on sequence homology to the calcium binding site in the factor XIII A chains (9).The tTG will selectively modify a group of protein-bound glutamine residues that exist in proteins found in the extracellular matrix (ECM) including vitronectin, fibronectin, osteonectin, and nidogen (1, 2). When tTG is released into plasma or ECM it binds to fibronectin and retains TGase activity (1, 2). Fibronectin binding functions to localize tTG to sites of fibronectin expression and deposition and limits the availability of the enzyme for cross-linking other substrates. The fibronectin binding site is located in the N-termi...

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Section: Resultsmentioning

confidence: 99%

Section: Quantitation Of Atp Hydrolysis and Analysis Of Reaction Prodmentioning

confidence: 99%

C-terminal Deletion of Human Tissue Transglutaminase Enhances Magnesium-dependent GTP/ATPase Activity

Lai

Slaughter

Koropchak

et al. 1996

Journal of Biological Chemistry

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“…Arginine has been extensively used to enhance refolding of proteins [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]. Its discover as a refolding assisting agent is rather surprising, considering its inhibitory effects on enzymatic activities [66,67].…”

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confidence: 99%

“…Since this enzyme is specific for arginine, one might expect that the effect is protein-specific. It turned out that the effect is non-specific and broad [50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65]69,70]. Arginine showed such effects on numerous proteins, including those that have resisted different refolding approaches.…”

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confidence: 99%

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

2012

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Monoclonal antibodies to the soluble antigens or cell surface markers hold great promise as effective human therapeutics. One of the major disadvantages is its large size, which prevents efficient penetration into the target tissues. Smaller version of antibodies, which has only antigen binding sites, is extensively investigated. It becomes increasingly apparent, however, that these smaller fragments of antibodies are rather difficult to produce, as the normally efficient mammalian secretion system does not work well for these fragments. Thus, refolding of insoluble proteins produced in Escherichia coli is a method of choice, although such refolding is mainly based on trial-and-error experiment. Here we describe a novel refolding system using a new amino acid-based detergent, N-lauroyl-L-glutamate, and arginine. This detergent appears to readily dissociate from proteins below critical micelle concentration (CMC), while remaining effective in protein solubilization above CMC. Arginine suppresses protein aggregation when the detergent concentration was reduced below CMC. The interaction of the detergent and arginine with proteins, which play an important role in protein refolding, will be discussed in great length.

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“…Protein modeling and biochemical studies have revealed, in great detail, the structure of tissue transglutaminase, indicating that the GTP-stabilized state is quite similar to the ligand-free enzyme [6,7]. This wealth of information on the structure of transglutaminase is removed from that on protein folding and stability to denaturation [8,9], with the exception of factor XIII, the structural perturbations of which during guanidine [10] and thermal treatment have been explored [11,12]. This information on tissue transglutaminase could be used in studies on protein folding and for its application to biotechnological projects.…”

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confidence: 99%

Conformational stability of human erythrocyte transglutaminase

Bergamini

Dean

Matteucci

et al. 1999

European Journal of Biochemistry

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Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0±9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.

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Denaturation-Renaturation of the Fibrin-Stabilizing Factor XIII a-Chain Isolated from Human Placenta. Properties of the Native and Reconstituted Protein

Cited by 25 publications

References 14 publications

C-terminal Deletion of Human Tissue Transglutaminase Enhances Magnesium-dependent GTP/ATPase Activity

C-terminal Deletion of Human Tissue Transglutaminase Enhances Magnesium-dependent GTP/ATPase Activity

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

Conformational stability of human erythrocyte transglutaminase

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