Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium <i>Thermotoga maritima</i>

Dams, Thomas; Ostendorp, Ralf; Ott, Markus; Rutkat, Kerstin; Jaenicke, Rainer

doi:10.1111/j.1432-1033.1996.0274h.x

Cited by 24 publications

(21 citation statements)

References 34 publications

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“…Hyperthermophilic phosphoribosylanthranilate isomerase is dimeric, but the proteins from mesophilic organisms are monomeric (7). Hyperthermophilic lactate dehydrogenase exists as tetrameric or octameric forms (8). Moreover, extra ion pairs or hydrophobic interactions have often been found in the subunit/subunit interface of proteins from hyperthermophiles (9 -18).…”

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confidence: 99%

Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability

Ogasahara¹,

Ishida²

2003

Journal of Biological Chemistry

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Tryptophan synthase from hyperthermophile, Pyrococcus furiosus, was found to be a tetrameric form (␣ 2 ␤ 2 ) composed of ␣ and ␤ 2 subunits. To elucidate the relationship between the features of the subunit association and the thermal stability of the tryptophan synthase, the subunit association and thermal stability were examined by isothermal titration calorimetry and differential scanning calorimetry, respectively, in comparison with those of the counterpart from Escherichia coli. The association constants between the ␣ and ␤ subunits in the hyperthermophile protein were of the order of 10 8 M ؊1 , which were higher by two orders of magnitude than those in the mesophile one. The negative values of the heat capacity change and enthalpy change upon the subunit association were much lower in the hyperthermophile protein than in the mesophile one, indicating that the conformational change of the hyperthermophile protein coupled to the subunit association is slight. The denaturation temperature of the ␣ subunit from the hyperthermophile was enhanced by 17°C due to the formation of the ␣ 2 ␤ 2 complex. This increment in denaturation temperature due to complex formation could be quantitatively estimated by the increase in the association constant compared with that of the counterpart from E. coli.Hyperthermophilic proteins, which retain the folded conformation and maximally express their function near the boiling point of water, have been the target of extensive studies on protein stabilization, folding, structure, and evolutionary aspects over the past decade. Much work has been done to determine the three-dimensional structures of hyperthermophile proteins and to identify the structural determinants of the enhanced stability. A comparison of the structures of proteins from hyperthermophiles with their mesophilic counterparts has led to a better understanding of several features of the hyperthermophile proteins (1, 2, 19 -22). One of these is that several hyperthermophile proteins have structures with a higher degree of oligomerization compared with the mesophilic homologues. Triose phosphate isomerase from hyperthermophiles is found to be tetrameric in contrast to the dimeric form from mesophilic sources (3-6). Hyperthermophilic phosphoribosylanthranilate isomerase is dimeric, but the proteins from mesophilic organisms are monomeric (7). Hyperthermophilic lactate dehydrogenase exists as tetrameric or octameric forms (8). Moreover, extra ion pairs or hydrophobic interactions have often been found in the subunit/subunit interface of proteins from hyperthermophiles (9 -18). On the bases of these observations, a hypothesis has been proposed that the higher order oligomerization of subunits and strong subunit association are potentially important for enhanced stability of hyperthermophile proteins (19 -22). However, there are few studies that characterize the strength of the subunit association in the hyperthermophile proteins and quantitatively elucidate the correlation between the subunit association and stability. E...

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mentioning

confidence: 99%

Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability

Ogasahara¹,

Ishida²

2003

Journal of Biological Chemistry

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“…Rabbit muscle LDH contains six Trp residues (47)(48)(49). In order to eliminate the Tyr disturbance, we detected intrinsic fluorescence of Trp residues of LDH at 292 nm, instead of 275 nm (41). Furthermore, neuronal tau (26) does not contain any Trp residue and its intrinsic fluorescence comes mostly from Tyr residues.…”

Section: Resultsmentioning

confidence: 99%

“…LDH (0.05 lM) was incubated with tau at different molar ratios (tau/LDH) at 80°C in the phosphate buffer (40,41) containing 1 mM DTT and 90 o light scattering (Ex ¼ Em ¼ 500 nm, 25°C) was measured.…”

Section: Methodsmentioning

confidence: 99%

“…According to Ma and Tsou (38), LDH (3 lM) was incubated in 3.0 M GdnHCl at 4°C for 16 h to reach the complete denaturation, and then the denatured enzyme was reactivated by dilution (25-fold) with the phosphate buffer containing tau of different concentrations and 1 mM DTT (40)(41)(42). The mixture was kept at 4°C for 1 h after dilution, followed by measurement of the activity (an aliquot of 50 ll) at different time intervals at 25°C.…”

Section: Methodsmentioning

confidence: 99%

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Chaperone-Like Manner of Human Neuronal Tau Towards Lactate Dehydrogenase

Tian

Nie

2004

Neurochem Res

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In our experiments, inactivation of lactate dehydrogenase (LDH, EC1.1.1.27) in the presence of human microtubule-associated tau is observably suppressed during thermal and guanidine hydrochloride (GdnHCl) denaturation. Kinetic studies show tau can prevent LDH from self-aggregation monitored by light scattering during thermal denaturation. On the other hand, neuronal tau promotes reactivation of LDH and suppresses self-aggregation of non-native LDH when GdnHCl solution is diluted. Furthermore, the reactivation yield of LDH decreases significantly with delayed addition of tau. All experiments were completed in the reducing buffer with 1 mM DTT to avoid between tau and LDH forming the covalent bonds during unfolding and refolding. Thus, Tau prevents proteins from misfolding and aggregating into insoluble, nonfunctional inclusions and assists them to refold to reach the stable native state by binding to the exposed hydrophobic patches on proteins instead of by forming or breaking covalent bonds. Additionally, tau remarkably enhances reactivation of GDH (glutamic dehydrogenase, EC 1.4.1.3), another carbohydrate metabolic enzyme, also showing a chaperone-like manner. It suggests that neuronal tau non-specifically functions a chaperone-like protein towards the enzymes of carbohydrate metabolism.

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“…LDH (50 M) was denatured completely by incubation in 3 M GdnHCl at 4°C overnight (16). Reactivation was initiated by a 50-fold dilution of the denatured enzyme into phosphate buffer containing different concentrations of PDI (or abbЈaЈ) and 10 mM DTT (17)(18)(19). The reactivation mixture was kept at 4°C for 30 min and then at 25°C for 2 h before an aliquot of 50 l was taken for activity assay at 25°C.…”

Section: Methodsmentioning

confidence: 99%

The Acidic C-terminal Domain Stabilizes the Chaperone Function of Protein Disulfide Isomerase

Tian

Wang³

et al. 2004

Journal of Biological Chemistry

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Protein disulfide isomerase (PDI, EC 5.3.4.1) is a chaperone and catalyzes the formation and rearrangement of disulfide bonds in proteins. Domain c-(463-491), containing 18 acidic residues, is an interesting and important C-terminal extension of PDI. In this study, the PDI mutant abba, in which domain c is truncated, was used to investigate the relationship between the C-terminal structure and chaperone function. Reactivation and light-scattering experiments show that both wild-type PDI and abba interact with lactate dehydrogenase (LDH, EC 1.1.1.27), which tends to self-aggregate during reactivation. The interaction enhances reactivation of LDH and reduces aggregation. According to these results, it seems as if domain c might be dispensable to the chaperone function of PDI. However, abba is prone to self-aggregation and causes increased aggregation of LDH during thermal denaturation. In contrast, wildtype PDI remains active as a chaperone under these conditions and prevents self-aggregation of LDH. Furthermore, measurements of intrinsic fluorescence and difference absorbance during denaturation show that abba is much more labile to heat or guanidine hydrochloride denaturation than wild-type PDI. This suggests that domain c is required for the stabilization and maintenance of the chaperone function of PDI under extreme conditions. Protein disulfide isomerase (PDI)1 contains 491 amino acid residues and is a multifunctional protein (1, 2). As an enzyme, it catalyzes the formation and rearrangement of disulfide bonds during oxidative protein folding. As a chaperone, PDI inhibits aggregation of the denatured protein and assists the refolding. The substrates of PDI can be proteins with or without disulfide bonds, such as D-glyceraldehyde-3-phosphate dehydrogenase (3), rhodanese (4), lysozyme (5), and acidic phospholipase A 2 (6). Under certain circumstances, PDI shows a so-called anti-chaperone effect in which substoichiometric concentrations of PDI facilitate aggregation and inhibit reactivation of denatured proteins (7), presumably by interacting and stabilizing the aggregates (8 -10).PDI consists of consecutive domains (a, b, bЈ, and aЈ) and the acidic C-terminal extension, domain c (8, 9, 11). Domains a and aЈ are similar to thioredoxin, and each contains the sequence CGHC, forming two independently acting catalytic sites (8, 11). Domains b and bЈ show low homology with domain a and have little sequence similarity to any member of the thioredoxin family but also adopt a thioredoxin fold (9). Domain c, also termed the C-terminal extension (residues 463-491), represents a putative Ca 2ϩ -binding region (11) and is rich in acidic amino acids. A C-terminal KDEL motif is necessary and sufficient for the retention of a polypeptide within the lumen of the endoplasmic reticulum (12). Koivunen et al. (10) have investigated the function of the PDI domain c using different mutants of PDI with deletions in the C-terminal sequence. They found that under routine conditions, domain c plays no significant role in the chaperone a...

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Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

Cited by 24 publications

References 34 publications

Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability

Stimulated Interaction between α and β Subunits of Tryptophan Synthase from Hyperthermophile Enhances Its Thermal Stability

Chaperone-Like Manner of Human Neuronal Tau Towards Lactate Dehydrogenase

The Acidic C-terminal Domain Stabilizes the Chaperone Function of Protein Disulfide Isomerase

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