The binding of 8-anilinonaphthalene-1-sulphonate to cytoplasmic aspartate aminotransferase from pig heart.
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ABSTRACT:
SUBMITTER: Harris HE
PROVIDER: S-EPMC1165195 | biostudies-other | 1975 Jan
REPOSITORIES: biostudies-other
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Project description:The unfolding of cytoplasmic aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from protein fluorescence, c.d. and thiol-group reactivity indicated that the enzyme was unfolded in 6 M-GdnHCl. Spectroscopic studies showed that this unfolding was accompanied by dissociation of the pyridoxal 5'-phosphate cofactor. On dilution of the GdnHCl, re-activation of the enzyme occurred in reasonable yield, provided that dithiothreitol and pyridoxal 5'-phosphate were present. The regain of activity obeyed second-order kinetics. In the absence of added dithiothreitol and pyridoxal 5'-phosphate, substantial formation of high-Mr aggregates occurred.
| S-EPMC1138799 | biostudies-other
Project description:Starch-gel electrophoresis of sheep heart aspartate aminotransferase was carried out over the range pH7.0-8.5. The enzyme separates into three subforms in the same way as the pig heart enzyme. As the pH was increased the distance migrated by each subform increased by the same amount, so that they remained the same distance apart. Titration of the enzyme over the appropriate pH range was used to calculate the difference in charge between the subforms and it was concluded that they differ by one charged group per dimer from their nearest neighbour on the electrophoretogram over the whole pH range studied. It was also shown that the pig-heart alpha and beta subforms differ by almost one charged group per dimer in the range pH5.5-5.7 and that the spacing between the subforms on starch-gel electrophoresis at pH8.0 is the same as that for the sheep enzyme. Since the charge difference between the subforms is maintained over such a wide range of pH, it is concluded that they probably differ from each other in covalent structure, because of the improbability that conformational differences can give rise to such behaviour. The relationship between the subforms and inactive binding of the coenzyme is also examined.
| S-EPMC1168092 | biostudies-other
Project description:The unfolding of the mitochondrial isoenzyme of aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) has been studied. By a number of criteria (enzyme activity, protein fluorescence, c.d., thiol-group reactivity), the enzyme was judged to be almost completely unfolded in 2 M-GdnHCl. On dilution of the GdnHCl, no re-activation of the enzyme could be observed, whether or not pyridoxal 5'-phosphate and dithiothreitol were present. The behaviour of the mitochondrial isoenzyme is in marked contrast with that of the cytoplasmic isoenzyme [West & Price (1989) Biochem. J. 261, 189-196], despite the similarities in the amino acid sequences and tertiary structures of the two isoenzymes. The implications of these findings for the process of folding and assembly of the mitochondrial isoenzyme in vivo are discussed.
| S-EPMC1136612 | biostudies-other
Project description:Fluorescence of 8-anilinonaphthalene-1-sulphonate is enhanced by both lactoferrin and transferrin. The enhancement is relatively low between pH 4 and 8, and high at below pH 4. At physiological pH the fluorescence enhancement is higher with the iron-deprived than with the iron-saturated proteins. Binding of iron by lactoferrin is associated with lowering affinity for the dye.
| S-EPMC1163837 | biostudies-other
Project description:Frailty is a common comorbidity associated with adverse events in patients with heart failure, and early recognition is key to improving its management. We hypothesized that the AST to ALT ratio (AAR) could be a marker of frailty in patients with heart failure. Data from the FRAGILE-HF study were analyzed. A total of 1327 patients aged ≥ 65 years hospitalized with heart failure were categorized into three groups based on their AAR at discharge: low AAR (AAR < 1.16, n = 434); middle AAR (1.16 ≤ AAR < 1.70, n = 487); high AAR (AAR ≥ 1.70, n = 406). The primary endpoint was one-year mortality. The association between AAR and physical function was also assessed. High AAR was associated with lower short physical performance battery and shorter 6-min walk distance, and these associations were independent of age and sex. Logistic regression analysis revealed that high AAR was an independent marker of physical frailty after adjustment for age, sex and body mass index. During follow-up, all-cause death occurred in 161 patients. After adjusting for confounding factors, high AAR was associated with all-cause death (low AAR vs. high AAR, hazard ratio: 1.57, 95% confidence interval, 1.02-2.42; P = 0.040). In conclusion, AAR is a marker of frailty and prognostic for all-cause mortality in older patients with heart failure.
| S-EPMC8184951 | biostudies-literature
Project description:BackgroundWe aimed to evaluate the association between the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio and subsequent development of any type of cancer in an apparently healthy population.MethodsWe conducted a retrospective cohort study at St. Luke's International Hospital, Tokyo, Japan between 2005 and 2018. All participants who visited for voluntary health checkups were included. We divided the participants into different quintiles based on the baseline AST/ALT ratios and examined the outcomes.ResultsA total of 85,658 participants were included. The mean age was 44.7 years (standard deviation 12.0) at baseline, and 42,913 (50.1%) of them were men. During a median follow-up of 61.6 months, 4701 (5.5%) participants developed some type of cancer. Compared with the middle AST/ALT ratio group, no other groups had similar adjusted hazard ratios (HR) for the development of any type of cancer in both men and women. When stratified by alcohol consumption, very high (adjusted HR 1.36; 95% CI 1.13-1.63) and high (adjusted HR 1.26; 95% CI 1.05-1.50) AST/ALT ratio groups among men who were regular drinkers had increased adjusted HRs for any type of cancer development, but the very high AST/ALT ratio group among men who were abstainers (adjusted HR 0.64; 95% CI 0.42-0.97) and very low AST/ALT ratio group among men who were occasional drinkers (adjusted HR 0.69; 95% CI 0.48-0.98) had lower adjusted HRs compared with the middle AST/ALT ratio group. Among women, regardless of alcohol consumption, adjusted HR for any type of cancer development was similar across all AST/ALT ratio groups.ConclusionPeople with higher AST/ALT ratios tended to have a higher risk of developing any type of cancer among men who were regular drinkers, but this risk was lower among men who were abstainers. Among women, regardless of alcohol consumption, there was no association between the development of any type of cancer and AST/ALT ratio.
| S-EPMC8817090 | biostudies-literature
Project description:Protein dynamics on various time scales from femtoseconds to milliseconds impacts biological function by driving proteins to conformations conducive to ligand binding and creating functional states in enzyme catalysis. Neutron vibrational spectroscopy carried out by measuring inelastic neutron scattering from protein molecules in combination with molecular simulations has the unique ability of detecting and visualizing changes in the picosecond protein vibrational dynamics due to ligand binding. Here we present neutron vibrational spectra of a homodimeric pyridoxal 5'-phosphate-dependent enzyme, aspartate aminotransferase, obtained from the open internal aldimine and closed external aldimine conformational states. We observe that in the external aldimine state the protein structure stiffens relative to the internal aldimine state, indicating rigidified vibrational dynamics on the picosecond time scale in the low-frequency regime of 5-50 cm-1. Our molecular dynamics simulations indicate substantial changes in the picosecond dynamics of the enzyme secondary structure elements upon substrate binding, with the largest contributions from just two helices and the ?-sheet.
| S-EPMC7408236 | biostudies-literature
Project description:1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.
| S-EPMC1165884 | biostudies-other
Project description:Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.
| S-EPMC7181641 | biostudies-literature
Project description:Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
| S-EPMC1177771 | biostudies-other