Purification and general properties of aspartate aminotransferase of ox heart.
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ABSTRACT:
SUBMITTER: Marino G
PROVIDER: S-EPMC1265045 | biostudies-other | 1966 Jun
REPOSITORIES: biostudies-other
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Project description:1. The amino acid composition of highly purified aspartate aminotransferase from ox heart was determined. 2. Alanine is the only N-terminal residue. 3. Leucine was identified as the only C-terminal residue. 4. No disulphide bridges are present in the enzyme molecule. 5. The thiol groups are not equally accessible, the accessibility being comparatively easier in the apoenzyme molecule.
| S-EPMC1265046 | biostudies-other
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:Aspartate aminotransferases (EC 2.6.1.1) catalyse the conversion of aspartate and alpha-ketoglutarate to oxaloacetate and glutamate in a reversible manner. Thus, the aspartate aminotransferase of Plasmodium falciparum (PfAspAT) plays a central role in the transamination of amino acids. Recent findings suggest that PfAspAT may also play a pivotal role in energy metabolism and the de novo biosynthesis of pyrimidines. While therapeutics based upon the inhibition of other proteins in these pathways are already used in the treatment of malaria, the advent of multidrug-resistant strains has limited their efficacy. The presence of PfAspAT in these pathways may offer additional opportunities for the development of novel therapeutics. In order to gain a deeper understanding of the function and role of PfAspAT, it has been expressed and purified to homogeneity. The successful crystallization of PfAspAT, the collection of a 2.8 A diffraction data set and initial attempts to solve the structure using molecular replacement are reported.
| S-EPMC2852332 | biostudies-literature
Project description:A recombinant version of a putative aspartate aminotransferase, AspB (encoded by the ORF Rv3565), from Mycobacterium tuberculosis (Mtb) was overexpressed in M. smegmatis and purified to homogeneity using liquid chromatography. Crystals of AspB were grown in a condition consisting of 0.2?M ammonium phosphate monobasic, 0.1?M calcium chloride dihydrate employing the hanging-drop vapour-diffusion method at 298?K. The crystals diffracted to a limit of 2.50?Å resolution and belonged to the orthorhombic space group P2?2?2?, with unit-cell parameters a=93.27, b=98.19, c=198.70?Å. The structure of AspB was solved by the molecular-replacement method using a putative aminotransferase from Silicibacter pomeroyi (PDB entry 3h14) as the search model. The template shares 46% amino-acid sequence identity with Mtb AspB. The crystal asymmetric unit contains four AspB molecules (the Mr of each is 42,035?Da).
| S-EPMC4089534 | biostudies-literature
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: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:BackgroundThe prognostic significance of the aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio in hepatocellular carcinoma remains uncertain. The aim of the current study was to evaluate the association between the AST/ALT ratio and prognosis in patients with hepatocellular carcinoma after hepatectomy, and to explore the role of underlying liver diseases as mediators.MethodsThis retrospective study included patients with hepatocellular carcinoma who underwent hepatectomy between January 2014 and January 2018 at two Chinese hospitals. The maximally selected rank statistic and g-computation approach were used to quantify and visualize the association between the AST/ALT ratio and overall survival or recurrence-free survival. The role of mediators (chronic hepatitis B, hepatic steatosis and liver cirrhosis) was analysed.ResultsAmong the 1519 patients (mean(s.d.) age at baseline, 50.5(11.3) years), 1309 (86.2%) were male. During a median follow-up of 46.0 months, 514 (33.8%) patients died and 358 (23.6%) patients experienced recurrence. The optimal cut-off value for the AST/ALT ratio was 1.4, and the AST/ALT ratio greater than or equal to 1.4 was independently associated with a 39.0% increased risk of death and a 30.0% increased risk of recurrence (overall survival: hazard ratio (HR), 1.39; 95% c.i. 1.15 to 1.68; recurrence-free survival: HR, 1.30; 95% c.i. 1.12 to 1.52) after adjusting for confounders. Chronic hepatitis B significantly mediated the association of the ratio of AST/ALT with both overall survival and recurrence-free survival (20.3% for overall survival; 20.1% for recurrence-free survival).ConclusionThe AST/ALT ratio greater than or equal to 1.4 was associated with shorter overall survival and recurrence-free survival in patients with hepatocellular carcinoma after hepatectomy, and chronic hepatitis B may play a role in their association.
| S-EPMC10798825 | biostudies-literature
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:Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549-561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a K(i) of 27 microm.
| S-EPMC95336 | biostudies-literature