Studies on the interaction of glutamate dehydrogenase with phospholipids and with mitrochondrial membranes

The object of this work was to characterise the interaction between beef liver glutamate dehydrogenase (EC 1.4.1.3), the inner mitochondrial membrane and its constituent phospholipids. Because of the similarities in the structure and properties of detergents with phospholipids, interaction of the enzyme with both types of amphiphiles was investigated. Interaction with phospholipid membranes was found to be a reversible process while detergents brought about irreversible denaturation at high concentrations. Association between the enzyme and the amphiphiles showed that the nature of the head group determines the extent of complex formation. Zwitter-ionic lysolecithin and phosphatidy1choline showed no interaction, while the anionic sodium dodecyl sulphate, phosphatidylserine and cardiolipin showed high affinity for binding to the enzyme. The apparent Ki values in the case of the two phospholipids were found to be 1 - 2 u M and 3 - 5 uM respectively in the direction of reductive aminatlon, The possibility of different conformations of the enzyme binding specifically to these charged surfaces and conformational changes brought about as a result of complex formation was investigated. The extent of binding decreased with increasing pHand ionic strength, suggesting contributions from electrostatic interactions. Evidence for hydrophobic interaction was indicated by the observation that the extent of complex formation increases with increasing temperature. Binding of the enzyme to mitochondrial membranes also indicated similar types of specificities. The enzyme showed a much higher affinity for binding to the inner surface of the inner mitochondrial membrane than to the outer surfaces of the inner and outer membranes. The extent of binding was also shown to depend on the presence of metabolites such as NADH and ADP. Binding decreased with increasing pH and ionic strength as was found for pure phospholipids. It is suggested that, in mitochondria, the reversible association between the enzyme and the inner mitochondrial membrane is controlled in a manner dependent upon the local pH, ionic strength and metabolite concentrations, and this may have important physiological significance in the control of metabolic activities of the enzyme. It is also suggested that the system exhibits the allosteric phenomenon which may be important for its regulation

A study on the two binding sites of hexokinase on brain mitochondria

<p>Abstract</p> <p>Background</p> <p>Type I hexokinase (HK-I) constitutes the predominant form of the enzyme in the brain, a major portion of which is associated with the outer mitochondrial membrane involving two sets of binding sites. In addition to the glucose-6-phosphate (G6P)-sensitive site (Type A), the enzyme is bound on a second set of sites (Type B) which are, while insensitive to G6P, totally releasable by use of high concentrations of chaotropic salts such as KSCN. Results obtained on release of HK-I from these "sites" suggested the possibility for the existence of distinct populations of the bound enzyme, differing in susceptibility to release by G6P.</p> <p>Results</p> <p>In the present study, the sensitivity of HK-I toward release by G6P (2 mM) and a low concentration of KSCN (45 mM) was investigated using rat brain, bovine brain and human brain mitochondria. Partial release from the G6P-insensitive site occurred without disruption of the mitochondrial membrane as a whole and as related to HK-I binding to the G6P-sensitive site. While, as expected, the sequential regime release-rebinding-release was observed on site A, no rebinding was detectable on site B, pre-treated with 45 mM KSCN. Also, no binding was detectable on mitochondria upon blocking site A for HK-I binding utilizing dicyclohexylcarbodiimide (DCCD), followed by subsequent treatment with KSCN. These observations while confirmed the previously-published results on the overall properties of the two sites, demonstrated for the first time that the reversible association of the enzyme on mitochondria is uniquely related to the Type A site.</p> <p>Conclusion</p> <p>Use of very low concentrations of KSCN at about 10% of the level previously reported to cause total release of HK-I from the G6P- insensitive site, caused partial release from this site in a reproducible manner. In contrast to site A, no rebinding of the enzyme takes place on site B, suggesting that site A is 'the only physiologically-important site in relation to the release-rebinding of the enzyme which occur in response to the energy requirements of the brain. Based on the results presented, a possible physiological role for distribution of the enzyme between the two sites on the mitochondrion is proposed.</p

Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein

How curcumin affords effective protection against amyloid fibrillation in insulin

Since the formation of amyloid structures from proteins was recognized in numerous diseases, many efforts have been devoted to the task of finding effective anti-amyloidogenic compounds. In a number of these investigations, the existence of generic compounds is implicitly acknowledged. Curcumin seems to be one of these compounds, possessing key structural components effective toward fibrillation prevention, and its anti-amyloidogenic property has been reported for a number of model and disease-related proteins such as lysozyme and alpha-synuclein. In this study, insulin amyloid formation has been shown to be effectively influenced by micromolar concentrations of curcumin. Under amyloidogenic conditions (pH 2.5 and 37 °C), the compound was observed to inhibit fibril formation of insulin in a dose-dependent manner. Moreover, addition of curcumin to the protein incubated under such conditions at different time points resulted in reduced amounts of final fibrils. Disaggregation of pre-formed fibrils was also observed upon addition of curcumin, as well as reduction in final fibril amounts after seeding. Overall, this compound appears to be able to interact with native, intermediate and fibrillar forms. Docking experiments suggest a potential interacting site with the B-chain of insulin, as well as the possibility for beta-sheet breaker activity. © 2013 The Royal Society of Chemistry

Chemical modification of glucose oxidase: possible formation of molten globule-like intermediate structure

AbstractChemical modification of lysine residues in glucose oxidase was carried out using citraconic anhydride. Modification brought about changes in the kinetic properties of the enzyme as evident by substantial lowering of Vmax and Km. Enhancement of tryptophan fluorescence was observed with a dramatic change in its pH dependence due to modification. Near- and far-UV circular dichroism spectra of the native and modified forms suggested formation of molten globule-like structures, further supported by 8-anilino-1-naphthalenesulfonic acid fluorescence which indicated higher exposure of hydrophobic residues as a result of chemical modification

Role of surface oxygen-containing functional groups of graphene oxide quantum dots on amyloid fibrillation of two model proteins.

There are many reports demonstrating that various derivatives of carbon nanoparticles are effective inhibitors of protein aggregation. As surface structural features of nanoparticles play a key role on modulating amyloid fibrillation process, in the present in vitro study, bovine insulin and hen egg white lysozyme (HEWL) were selected as two model proteins to investigate the reducing effect of graphene oxide quantum dots (GOQDs) on their assembly under amyloidogenic conditions. GOQDs were prepared through direct pyrolysis of citric acid, and the reduction step was carried out using ascorbic acid. The prepared nanoparticles were characterized by UV-Vis, X-ray photoelectron, and FT-IR spectroscopies, transmission electron and atomic force microscopies, zeta potential measurement, and Nile red fluorescence assay. They showed the tendencies to modulate the assembly of the proteins through different mechanisms. While GOQDs appeared to have the capacity to inhibit fibrillation, the presence of reduced GOQDs (rGOQDs) was found to promote protein assembly via shortening the nucleation phase, as suggested by ThT fluorescence data. Moreover, the structures produced in the presence of GOQDs or rGOQDs were totally nontoxic. We suggest that surface properties of these particles may be part of the differences in their mechanism(s) of action

Inhibition of HEWL fibril formation by taxifolin: Mechanism of action

<div><p>Among therapeutic approaches for amyloid-related diseases, attention has recently turned to the use of natural products as effective anti-aggregation compounds. Although a wealth of <i>in vitro</i> and <i>in vivo</i> evidence indicates some common inhibitory activity of these compounds, they don’t generally suggest the same mechanism of action. Here, we show that taxifolin, a ubiquitous bioactive constituent of foods and herbs, inhibits formation of HEWL amyloid fibrils and their related toxicity by causing formation of very large globular, chain-like aggregates. A range of amyloid-specific techniques were employed to characterize this process. We found that taxifolin exerts its effect by binding to HEWL prefibrillar species, rather than by stabilizing the molecule in its native-like state. Furthermore, it’s binding results in diverting the amyloid pathway toward formation of very large globular, chain-like aggregates with low β-sheet content and reduced solvent-exposed hydrophobic patches. ThT fluorescence measurements show that the binding capacity of taxifolin is significantly reduced, upon generation of large protofibrillar aggregates at the end of growth phase. We believe these results may help design promising inhibitors of protein aggregation for amyloid-related diseases.</p></div