Removal of an N-terminal peptide from mitochondrial aspartate aminotransferase abolishes its interactions with mitochondria in vitro.
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ABSTRACT:
SUBMITTER: O'Donovan KM
PROVIDER: S-EPMC1145029 | biostudies-other | 1985 Jun
REPOSITORIES: biostudies-other
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Project description:Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.
| S-EPMC3046307 | biostudies-literature
Project description:Cobalt nanoparticles (Co-NPs) have been extensively used in clinical practices and medical diagnosis. In this study, the potential toxicity effects of Co-NPs with special emphasis over the biochemical enzyme activities, such as aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in serum, liver, and kidney of Wistar rats were investigated. This toxicity measurement of nanomaterials can support the toxicological data. The biochemical enzymatic variations are powerful tools for the assessment of toxicity. ASAT and ALAT enzymes have been widely used to predict tissue-specific toxicities associated with xenobiotic. The biochemical changes induced by Co-NPs have significance in their toxicological studies because the alterations in biochemical parameters before clinical symptoms indicate either their toxicant safety or detrimental effect. Herein, Co-NPs with particle size <50 nm significantly activated ASAT and ALAT enzymes in the serum, liver, and kidney of rats at concentration-dependent order.
| S-EPMC7184135 | biostudies-literature
Project description:1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.
| S-EPMC1158118 | biostudies-other
Project description:Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.
| S-EPMC1145008 | biostudies-other
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:1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.
| S-EPMC1198859 | biostudies-other
Project description:Mitochondrial aspartate aminotransferase is inactivated irreversibly on heating. The inactivated protein aggregates, but aggregation is prevented by the presence of the chaperonin 60 from Escherichia coli (GroEL). The chaperonin increases the rate of thermal inactivation in the temperature range 55-65 degrees C but not at lower temperatures. It has previously been shown [Twomey and Doonan (1997) Biochim. Biophys. Acta 1342, 37-44] that the enzyme switches to a modified, but catalytically active, conformation at approx. 55-60 degrees C and the present results show that this conformation is recognized by and binds to GroEL. The thermally inactivated protein can be released from GroEL in an active form by the addition of chaperonin 10 from E. coli (GroES)/ATP, showing that inactivation is not the result of irreversible chemical changes. These results suggest that the irreversibility of thermal inactivation is due to the formation of an altered conformation with a high kinetic barrier to refolding rather than to any covalent changes. In the absence of chaperonin the unfolded molecules aggregate but this is a consequence, rather than the cause, of irreversible inactivation.
| S-EPMC1219682 | biostudies-other
Project description:The kinetic behaviour of chicken liver and turkey liver aspartate aminotransferases (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was studied. Steady-state data were obtained from a wide range of concentrations of substrates and product L-glutamate. The data were fitted by rational functions of degree 1:1, 1:2 and 2:2 with respect to substrates and 0:1, 1:1, 0:2 and 1:2 with regard to product (L-glutamate), by using a non-linear regression program that guarantees the fit. The goodness of fit was improved by the use of a computer program that combines model discrimination parameter refinement and sequential experimental design. It was concluded that aspartate aminotransferase requires a minimum velocity equation of degree 2:2 for L-aspartate, 2:2 for 2-oxoglutarate and 1:2 for L-glutamate. Finally, a plausible kinetic mechanism that justifies these experimental results is proposed.
| S-EPMC1148927 | biostudies-other
Project description:Rat liver mitochondrial aspartate aminotransferase (a homodimer) was shown to catalyse a beta-lyase reaction with three nephrotoxic halogenated cysteine S-conjugates [ S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(2-chloro-1,1,2-trifluoroethyl)-L-cysteine], and less effectively so with a non-toxic cysteine S-conjugate [benzothiazolyl-L-cysteine]. Transamination competes with the beta-lyase reaction, but is not favourable. The ratio of beta elimination to transamination in the presence of S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and 2-oxoglutarate is >100. Syncatalytic inactivation by the halogenated cysteine S-conjugates is also observed. The enzyme turns over approx. 2700 molecules of halogenated cysteine S-conjugate on average for every monomer inactivated. Kidney mitochondria are known to be especially sensitive to toxic halogenated cysteine S-conjugates. Evidence is presented that 15-20% of the cysteine S-conjugate beta-lyase activity towards S -(1,1,2,2-tetrafluoroethyl)-L-cysteine in crude kidney mitochondrial homogenates is due to mitochondrial aspartate aminotransferase. The possible involvement of mitochondrial aspartate aminotransferase in the toxicity of halogenated cysteine S-conjugates is also discussed.
| S-EPMC1222959 | biostudies-other