Thermal stability of α-glycerophosphate dehydrogenase in Drosophila

Thermal stability of -glycerophosphate dehydrogenase-1 (-Gpdh-1) in nine Drosophila species was studied at pH's ranging from 6.4 to 8.5. This was done by measuring the changes in the activity of enzymes during the heat denaturation process. In addition to temperature, the rate of denaturation is highly dependent on the pH of the incubation buffer. The results of this study show that the thermal stability of enzyme molecules is different in different species. This holds true also in the species in which the enzymes have been found to be identical by other means. The differences between species of the Drosophila virilis group are discussed.This study was supported by funds from the National Research Council of Sciences of Finland.     

Thermal adaptation of α-amylases: a review

The temperature adaptation of α-amylase can be gained by different adjustments in protein structure with consecutive effects on the stability and flexibility of the protein. In this review, meso, thermo and cold-active α-amylases have been compared with respect to their structure and intramolecular interactions. With decrease in temperature, the number of ionic interactions also decreases, leading to greater flexibility of proteins. It has also been observed that the proline and arginine content is higher in thermophilic amylases as compared to meso and psychrophilic amylases, increasing the rigidity and structural stability of protein molecule.     

Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin

Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 3.4.21.1) or the aspartic protease, pepsin (EC 3.4.23.1). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.     

Complete nucleotide sequence of a chicken α-globin cDNA

The entire sequence of a 541 bp insert in recombinant plasmid pHb1003 has been determined. This plasmid, which was shown to carry a cloned cDNA copy of the chicken α-globin mRNA, contains the complete structural gene as well as 19 bp of the 5'-untranslated region and 99 bp of the 3'-untranslated region. This sequence may encode a non-adult α-globin gene, especially since the cDNA clones were generated from phenylhydrazine-induced, globin-specific mRNA extracted from anemic white leghorns. The possibility that this α-globin might represent a stress globin is considered.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Thermal stability of immobilized α-chymotrypsin is additionally increased by chaotropic compounds

Summary Temperature dependence of the rate constant of irreversible thermal inactivation, k_in, of immobilized -chymotrypsin depends markedly on the number of covalent bonds between the enzyme and support. When the number of bonds is big enough (thirteen), the dependence is linear as presented in Arrhenius plot (log k_in versus reciprocal temperature). However, if the number of such bonds is moderate or small (six or two), the temperature dependence of k_in, has a pronounced zig-zag character. This difference in the inactivation behaviour is attributed to an ability of moderately or mildly attached -chymotrypsins to accomplish a transition into a less ordered, catalytically inactive conformation and to inability of rigidly bound enzyme to pass such a transition. Chaotropic salts additionally stabilize this loose conformation of mildly or moderately bound -chymotrypsins against irreversible thermal inactivation but are without effect on the stability of rigidly bound enzyme.     

Separation and properties of a “Neutral” hexosaminidase from embryonic chicken brain

• 1.1. A “neutral” hexosaminidase has been separated from other hexosaminidase forms (I and II) by DEAE-cellulose chromatography and characterized in embryonic (16-days old) and 1-day old chicken brains.
• 2.2. Its properties differ from those of the forms I and II. It has optimum activity at about pH 6.0 and can be eluted from DEAE-cellulose with 0.25 M KCl only.
• 3.3. It has no N-acetylgalactosaminidase activity and cannot be successfully detected after isoelectric focusing since it is very acidic and completely unstable below pH 5.0.
• 4.4. “Neutral” hexosaminidase is heat-stable at pH 6.0 and is inhibited by chloride.
• 5.5. These properties, very different from those of forms I and II, suggest that this “neutral” form of hexosaminidase would be very similar to known hexosaminidase C separated from other materials.
• 6.6. We have found no significant differences for the above-mentioned three forms in chick embryos (16-days old) in comparison with those from 1-day old chicken.

     

The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response

α-Synuclein (Snca) is an abundant small cytosolic protein (140 amino acids) that is expressed in the brain, although its physiological role is poorly defined. Consistent with its ubiquitous distribution in the brain, we and others have established a role for Snca in brain lipid metabolism and downstream events such as neuroinflammation. In astrocytes, Snca is important for fatty acid uptake and trafficking, where its deletion decreases 16:0 and 20:4n-6 uptake and alters targeting to specific lipid pools. Although Snca has no impact on 22:6n-3 uptake into astrocytes, it is important for its targeting to lipid pools. Similar results for fatty acid uptake from the plasma are seen in studies using whole mice coupled with steady-state kinetic modeling. We demonstrate in gene-ablated mice a significant reduction in the incorporation rate of 20:4n-6 into brain phospholipid pools due to reduced recycling of 20:4n-6 through the ER-localized long-chain acyl-CoA synthetases (Acsl). This reduction results in a compensatory increase in the incorporation rate of 22:6n-3 into brain phospholipids. Snca is also important for brain and astrocyte cholesterol metabolism, where its deletion results in an elevation of cholesterol and cholesteryl esters. This increase may be due to the interaction of Snca with membrane-bound enzymes involved in lipid metabolism such as Acsl. Snca is critical in modulating brain prostanoid formation and microglial activities. In the absence of Snca, microglia are basally activated and demonstrate increased proinflammatory cytokine secretion. Thus, Snca, through its modulation of brain lipid metabolism, has a critical role in brain inflammatory responses.     

Thermal stability of carbon [n,5] prismanes (n = 2–4): a molecular dynamics study

Tight-binding molecular dynamics simulations are carried out to analyse the thermal stability of the carbon [n,5] prismanes with n = 2–4 over a wide temperature range. The results obtained demonstrate that the isomerisation activation energy as well as the frequency factor in the Arrhenius equation of these metastable nanostructures rapidly decreases with an increase of n. Therefore, the increase in the effective length of [n,5] prismane leads to the decrease in its lifetime up to the moment of its isomerisation. Nevertheless, the stability of [n,5] prismanes is confirmed to be sufficient for their existence at the liquid-nitrogen temperature. The main identified mechanism of [n,5] prismanes isomerisation is the interlayer C–C bond breaking leading to their transformation to the hypostrophene-based molecular systems.     

S′-subsite mapping of polyethyelene glycol-modified α-chymotrypsin and α-chymotrypsin: a comparative study

Nucleophile specificities of polyethylene glycol-modified α-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amines both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P′₁ position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P′₂ were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P′₁. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P′₁ > P′₃ >P′₂; this is valid for both enzymes.     

Thermal stability of cytochrome c₅ of pressure-sensitive Shewanella livingstonensis

Cytochrome c? of pressure-sensitive Shewanella livingstonensis (SL cytc?) exhibits lower thermal stability than a highly homologous counterpart of pressure-tolerant Shewanella violacea. This stability difference is due to an enthalpic effect that can be attributed to the amino acid residue at position 50 (Leu or Lys). These cytc? proteins are appropriate materials for understanding the protein stability mechanism.     

Cloning and characterization of chicken α5 integrin: Endogenous and experimental expression in early chicken embryos

Key roles for fibronectin and its integrin receptors have been postulated in the multiple cell-matrix interactions essential for chick embryo morphogenesis. However, mechanistic studies of these processes have been hampered by the current absence of sequence data and chicken cDNA clones for the major fibronectin receptor subunit, integrin α5 (ITGA5). We report here the sequence, endogenous expression pattern, and transfection of full-length chicken integrin α5. During early chicken embryonic development, α5 is highly expressed in cranial neural folds and migrating neural crest cells, suggesting potential roles in neural crest formation and migration. In fact, over-expression of this integrin in early neural tube selectively induces BMP5, a growth factor recently implicated in neural crest formation. Availability of these α5 integrin tools should facilitate studies of its functions in early embryonic development.     

Biochemical genetics of α-amylase isozymes of the chicken pancreas

In the chicken population at large, three electrophoretically distinct pancreatic alpha-amylase isozymes were discovered. The isozymes were designated Pa 1, Pa 2, and Pa 3. The local population of chickens, however, possessed only isozymes Pa 2 and Pa 3 present as three phenotypes: Amy-2 B, consisting of isozyme Pa2; Amy2 BC, consisting of isozymes Pa 2 plus Pa 3; and Amy2 C, consisting of isozyme Pa 3. Pancreatic biopsy permitted the establishment of a breeding flock with defined amylase phenotypes. Matings of this flock established that amylases are inherited as codominant alleles at a single genetic locus. Further, there was no evidence of ontogenetic modification of the amylase isozymes. It was observed that amylase isozymes Pa 2 and Pa 3 each generated a family of at least three faster-migrating amylolytic proteins. These post-translationally modified amylases were designated Pa Xa, Pa Xb, and Pa Xc, where X represents the number of the progenitor amylase. Structural analyses of purified amylases demonstrated that all amylase isozymes are nonglycosidated, monomeric molecules of molecular weight 55,000. In addition, the data are consistent with the hypothesis that the faster-migrating amylases are produced by deamidation of asparagine and/or glutamine residues.     

The study of the brain’s organization of creativity: I. Development of a psychological test

This article describes an original psychological test on verbal creative work comprising a number of tasks designed to study the organization of creative processes in human brain with the help of positron emission tomography (PET) and electroencephalography (EEG). When approved in a group of 30 healthy volunteers, the test revealed principal cognitive strategies of performing creative tasks which formed the basis for elaborating criteria for selecting subjects for physiological study. Empirical substantiation of the proposed test showed its validity for the use in the psychophysiological investigation of creative work.     

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal β-glycosidases

Sβgly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of Sβgly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure–function relationships in these enzymes. Received: May 20, 1999 / Accepted: January 10, 2000     

Thermal stability of tropocollagens--are hydrogen bonds really important?

The variation in thermal stability of tropocollagens from different species is known to be related to the total pyrrolidine content of the collagens. Thermodynamic analysis of the available data gives results that are inconsistent with the hypothesis that inter-chain hydrogen bonds are the major source of stability in the tropocollagen triple helix.     

Metabolic fate of a high concentration of glutamine and glutamate in rat brain slices: a ¹³C NMR study

This study was performed to analyze the metabolic fate of a high concentration (5 mM) of glutamine and glutamate in rat brain slices and the participation of these amino acids in the glutamine-glutamate cycle. For this, brain slices were incubated for 60 min with [3-13C]glutamine or [3-13C]glutamate. Tissue plus medium extracts were analyzed by enzymatic and 13C NMR measurements and fluxes through pathways of glutamine and glutamate metabolism were calculated. We demonstrate that both substrates were utilized and oxidized at high rates by rat brain slices and served as precursors of neurotransmitters, tricarboxylic acid (TCA) cycle intermediates and alanine. In order to determine the participation of glutamine synthetase in the appearance of new glutamine molecules with glutamine as substrate, brain slices were incubated with [3-13C]glutamine in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase. Our results indicate that 36.5% of the new glutamine appeared was glutamine synthetase-dependent and 63.5% was formed from endogenous substrates. Flux through glutamic acid decarboxylase was higher with glutamine than with glutamate as substrate whereas fluxes from α-ketoglutarate to glutamate and through glutamine synthetase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase were in the same range with both substrates.     

Inherited haemolytic anaemia created by insertional inactivation of the α-spectrin gene

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. Byin situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the α-spectrin gene locus.