Enhancement of L-3-hydroxybutyryl-CoA dehydrogenase activity and circulating ketone body levels by pantethine. Relevance to dopaminergic injury.

BACKGROUND: The administration of the ketone bodies hydroxybutyrate and acetoacetate is known to exert a protective effect against metabolic disorders associated with cerebral pathologies. This suggests that the enhancement of their endogenous production might be a rational therapeutic approach. Ketone bodies are generated by fatty acid beta-oxidation, a process involving a mitochondrial oxido-reductase superfamily, with fatty acid-CoA thioesters as substrates. In this report, emphasis is on the penultimate step of the process, i.e. L-3-hydroxybutyryl-CoA dehydrogenase activity. We determined changes in enzyme activity and in circulating ketone body levels in the MPTP mouse model of Parkinson's disease. Since the active moiety of CoA is pantetheine, mice were treated with pantethine, its naturally-occurring form. Pantethine has the advantage of being known as an anti-inflammatory and hypolipidemic agent with very few side effects. RESULTS: We found that dehydrogenase activity and circulating ketone body levels were drastically reduced by the neurotoxin MPTP, whereas treatment with pantethine overcame these adverse effects. Pantethine prevented dopaminergic neuron loss and motility disorders. In vivo and in vitro experiments showed that the protection was associated with enhancement of glutathione (GSH) production as well as restoration of respiratory chain complex I activity and mitochondrial ATP levels. Remarkably, pantethine treatment boosted the circulating ketone body levels in MPTP-intoxicated mice, but not in normal animals. CONCLUSIONS: These finding demonstrate the feasibility of the enhancement of endogenous ketone body production and provide a promising therapeutic approach to Parkinson's disease as well as, conceivably, to other neurodegenerative disorders.

[The protective effects of Zn-metallothionein on severely scalded rats inflicted by oxygen free radicals].

OBJECTIVE: To investigate the protective effects of metallothionein (MT) on severely scalded rats inflicted by oxygen free radicals after delayed resuscitation. METHODS: Wistar rats inflicted by 30% TBSA III degree scalding injury were taken as the model. Fifty-Six rats were randomly divided into four groups, i.e. normal control, delayed resuscitation, MT treated and VitC treated groups. The changes in superoxide dismutase (SOD) and malondialdehyde (MDA) contents in plasma and subeschar edematous fluid were determined at 24 and 48 postburn hours with traditional indirect detecting method and electron spin resonance (ESR). Pathomorphological examination was simultaneously carried out for cardiac, hepatic, renal and intestinal samples. Plasma biochemical indices were determined at the same time. RESULTS: In delayed resuscitation group, SOD content decreased and the MDA contents increased in plasma and subeschar fluid obviously, with remarkable changes in the pathomorphology of all the internal organs and the blood biochemical indices. But in MT treating group, SOD content increased (P < 0.05) with decreased content of MDA (P < 0.05) and the pathomorphology and blood biochemical indices improved significantly when compared with those in delayed resuscitation group and VitC treatment group. CONCLUSION: MT treatment might be beneficial in the management of severely scalding rats infliced by oxygen free radicals.

Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters). Six cysteine mutants each have an increased K(m)(NADH) (2-6-fold) but an unchanged K(m)(NAD)+. The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site. Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.

Favourable, significant effect of the dose-dependent treatment with RU 38486 (RU) on the alterations of the hepatic mitochondrial function of diabetic rats.

In the present work, the effect "in vivo' of increasing doses of RU 38486 upon the hepatic mitochondrial function of diabetic rats has been studied. At the same time, the action of adrenalectomy and corticosterone restitution on this function were comparatively demonstrated. The parameters measured were oxygen consumption with the substrates: 3-hydroxybutyrate (HB), succinate (Suc) and malate-glutamate (Mal-glut) in intact liver mitochondria and the activities of 3-hydroxybutyrate dehydrogenase (HBD) and cytochrome c oxidase (Cyt.c oxid.) enzymes in broken liver mitochondria. The groups of animals studied were normal controls (N) and the following groups of diabetic rats: rats without any treatment (D), adrenalectomized rats (D+ADX), rats that were adrenalectomized and treated with corticosterone (D+ADX+C) and four groups treated with increasing oral doses of RU (in mg/kg body wt.), that is, 12.5 (D+RU1), 25.0 (D+RU2), 37.5 (D+RU3) and 50.0 (D+RU4). The results showed a tendency of increasing values of mitochondrial oxygen consumption in diabetic animals treated with RU. The favourable effect of increasing doses of RU on O2 consumption of diabetic rat liver mitochondria with each of the substrates showed a significant association as indicated by the values obtained for the correlation coefficients r (0.95, 0.97 and 0.99 according to the substrate HB, Succ or Mal-glut, respectively). Likewise, the correlation between the treatment with increasing doses of RU and the recovery of enzyme activities showed a significant dose-effect association with r 0.94 for HBD and r = 0.95 for Cyt.c oxid. Adrenalectomy showed a similar effect to treatment with the maximum dose of RU while corticosterone restitution gave measured values similar to those of the D group. In conclusion, the favourable, significant variation of the hepatic mitochondrial function of diabetic rats was demonstrated by the dose-dependent treatment with RU as seen by the correlation statistical study performed. At the same time, the pernicious effect that glucocorticoids exert upon such function in experimental diabetes was confirmed.

Effect of peroxisomes proliferators and hypolipemic agents on mitochondrial inner membrane linked D-3-hydroxybutyrate dehydrogenase (BDH).

1. D-3-hydroxybutyrate dehydrogenase EC 1.1.1.30 (BDH) activity was measured in mitochondria of rats submitted to an intermittent feeding treatment with ciprofibrate or fenofibrate, i.e. fibrate analogues with hypolipemic activity and peroxisome proliferation properties. Our data shows an inhibition of rat liver mitochondrial BDH activity. This inhibitory effect is abolished when the treatment is stopped and reappears after a second treatment. 2. Incubation of hypolipemic agents (ciprofibrate, clofibrate, clobuzarit, fenofibrate or 2,4 dichlorophenoxyacetic acid) with submitochondrial linked BDH leads to an inhibition in a concentration dependent manner. 3. The protection by NAD(H) (coenzymes) and by methyl-malonate (a substrate analogue and competitive inhibitor) indicates that the inhibition occurs in the active site. On the other hand, there is a strong protection by phospholipid vesicles. This trapping effect may be attributed to lipophilic properties of hypolipemic agents. 4. Comparative effect of hypolipemic agents on mitochondrial BDH activity from rat liver and from Tetrahymena pyriformis indicates the same inhibition and same protection effects. This supports conservation of the enzymatic properties according to the evolution.

Occurrence, purification and properties of the staphylococcal beta-hydroxybutyrate dehydrogenase.

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30.)-an enzyme involved in degradation of polymer store material-was found in staphylococci. The enzyme was isolated from Staphylococcus xylosus NCTC D100694 cells, purified and characterized. The native enzyme is a tetramer and consists of equal subunits. Its relative molecular mass is M(r) = 140 kDa and pI = 4.7. The enzyme activity is stimulated by Mg+2 and Ca+2 ions. Staphylococcal beta-hydroxybutyrate dehydrogenase is relatively stable and active in a wide temperature range. The optimum pH for oxidation is 8.6 and for reduction 6.7. The enzyme is highly specific for D(-)stereoisomer of beta-hydroxybutyrate. Km values for beta-hydroxybutyrate and acetoacetate are 39.1 microM and 5.47 microM, respectively.

[The activities of aldehyde dehydrogenase, GABA-aminotransferase and succinic semialdehyde reductase in the brain of rats with different preferences and tolerances for ethanol]. / Aktivnost' al'degiddegidrogenazy, GAMK-aminotransferazy i reduktazy iantarnogo polual'degida v mozge krys s razlichnym otnosheniem k étanolu i tolerantnost'iu k nemu.

The formation and catabolism of aldehydes were compared in the hemispheres and brain stem of rats preferring ethanol (EP) or water (WP) and of those which were high tolerant (HT) and low tolerant to the hypnotic effect of ethanol. It was shown that aldehyde dehydrogenase was more active in the brain stem of HT-EP rats than that of HT-WR or LT-EP animals, whereas GABA aminotransferase is most active in the hemispheres and brain stem of LT-EP rats. The total activity of succinic semialdehyde reductase was equal in all the groups studied; however kinetic analysis suggest that the enzyme has a higher affinity for the substrate and coenzyme in the brain stem of HT-EP rats. Ethanol administered to HT-EP animals suppressed aldehyde dehydrogenase in the brain stem, unchanged GABA aminotransferase and activates succinic semialdehyde reductase in the two brain structures.

Phospholipid polar head specificity of D-3-hydroxybutyrate dehydrogenase activation studied by new synthetic phospholipids and analogues.

D-3-hydroxybutyrate dehydrogenase, an inner-mitochondrial enzyme responsible for the interconversion of two ketone bodies, is a well known phospholipid dependent enzyme. Newly synthesized phospholipid analogues were used to study the structural requirement for lipid activation of the purified enzyme. A positive charge on the polar head is required but must be at the surface of lipid vesicles. In contrast the maximum velocity and the Michaelis constant values are not strongly dependent on the nature of the zwitterionic phospholipid polar head.