Slawomir Strumilo

Up-regulation of 2-oxoglutarate dehydrogenase as a stress response

2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca(2+)- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl(2)), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Kinetic and spectral investigation of allosteric interaction of coenzymes with 2-oxo acid dehydrogenase complexes

The possible role of thiamine pyrophosphate (TPP) in the regulation of both multienzyme pyruvate dehydrogenase complex (PDC) and 2-oxoglutarate dehydrogenase complex (OGDC) has been investigated by kinetic and spectral methods. The purified PDC and OGDC from animal heart muscle were near saturated with endogenous TPP. The PDC containing the bound coenzyme showed hysteretic behaviour manifested in a lag phase of the catalysed reaction after the contact of PDC with substrates. Exogenous TPP added to the full reaction medium led to a disappearance of the lag phase and to strong reduction of the Michaelis constant (Km) value for pyruvate, and more moderate decrease of Km for both coenzyme A and NAD. In the case of OGDC exogenous TPP also decreased S0.5 (Km) for substrate 2-oxoglutarate. In addition, exogenous TPP changed both the UV and circular dichroism spectra of PDC and last one of OGDC, and lowered the fluorescence emission of the multienzyme complexes containing bound molecules of endogenous coenzyme in their active sites. Thiamine pyrophosphate seems to play, besides its coenzyme function, the role of positive allosteric effector which causes conformational changes of the multienzyme complexes and increases their affinity to substrates. q 2002 Elsevier Science B.V. All rights reserved.

Often ignored facts about the control of the 2‐oxoglutarate dehydrogenase complex

Information about the control of the activity of the 2‐oxoglutarate dehydrogenase complex (OGDHC), a key enzyme in the citric acid cycle, is not well covered in the biochemical education literature, especially as it concerns the allosteric regulation of OGDHC by adenine nucleotide and ortophosphate. From experimental work published during the last 25 years, the following basic view is clear: (a) animal OGDHC is very sensitive to ADP, Pi, and Ca2+; (b) these positive effectors increase the affinity of OGDHC to 2‐oxoglutarate; (c) OGDHC is inhibited by ATP, NADH, and succinyl‐CoA; (d) the ATP effect is realized mainly via opposition to ADP activation; (e) NADH, in addition to inhibiting the dihydrolipoamide dehydrogenase component of the enzyme complex (competitively versus NAD+) decreases the affinity of 2‐oxoglutarate dehydrogenase to its substrate; (f) bacterial and plant OGDHC are activated by AMP instead of ADP. These main effects form the basis of short term regulation of OGDHC. It is desirable that such information should reach biochemistry students.

Comparison of properties of malate dehydrogenase isoenzymes from hare and rabbit hearts

The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct reaction) and oxaloacetate and NADH (in reverse reaction), i.e., they have lower KM values in comparison with the isoenzymes from the rabbit heart. Malate dehydrogenase seems to operate more effectively in the hare heart, which might be important in adaptive and evolutionary aspects.

Differences in some properties of lactate dehydrogenase from muscles of the carp Cyprinus carpio and trout Salmo gairdneri

Some catalytic and kinetic properties of lactate dehydrogenase (LDH) isolated from trout and carp skeletal muscles were compared. The specific activity of LDH in the carp muscle was lower by about one third than the activity in the trout muscle. Temperature and pH optima for LDH isolated from the carp muscle were higher than those for the trout muscle LDH. Moreover, in direct reaction, the carp muscle LDH had a higher affinity both for pyruvate and for NADH, i.e., it had lower KM values. Instead, the trout muscle LDH showed the positive kinetic cooperativity (the Hill coefficient > 1) of the substrate and coenzyme binding sites. Thus, the carp LDH seems to function more effectively under anaerobic conditions and at higher temperatures.

Thiamine antivitamins – an opportunity of therapy of fungal infections caused by Malassezia pachydermatis and Candida albicans

Severe skin diseases and systemic fungaemia are caused by Malassezia pachydermatis and Candida albicans respectively. Antifungal therapies are less effective because of chronic character of infections and high percentage of relapses. Therefore, there is a great need to develop new strategies of antifungal therapies. We previously found that oxythiamine decreases proliferation of yeast (Saccharomyces cerevisiae), therefore we suggest that thiamine antivitamins can be considered as antifungal agents. The aim of this study was the comparison of thiamine antivitamins (oxythiamine, amprolium, thiochrome, tetrahydrothiamine and tetrahydrooxythiamine) inhibitory effect on the growth rate and energetic metabolism efficiency in non‐pathogenic S. cerevisiae and two potentially pathogenic species M. pachydermatis and C. albicans. Investigated species were cultured on a Sabouraud medium supplemented with trace elements in the presence (40 mg l−1) or absence of each tested antivitamins to estimate their influence on growth rate, enzyme activity and kinetic parameters of pyruvate decarboxylase and malate dehydrogenase of each tested species. Oxythiamine was the only antivitamin with antifungal potential. M. pachydermatis and S. cerevisiae were the most sensitive, whereas C. albicans was the least sensitive to oxythiamine action. Oxythiamine can be considered as supportive agent in superficial mycoses treatment, especially those caused by species from the genus Malassezia.

Kinetic properties of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes from a new source—the heart of wild boar Sus scrofa

the properties of PDHC (own data) and OGDHC (literature data) from the heart muscle of the domestic pig Sus scrofa f. domestica. The first step in isolation of the 2-oxoacid dehydrogenase complexes from the wild boar heart consisted in obtaining the myocardial mitochondrial fraction. This allowed disregarding a multitude of proteins localized elsewhere. Mitochondria were then destroyed by freezing in liquid nitrogen with subsequent thawing, while PDHC and OGDHC were precipitated from the centrifuged mitochondrial extract by polyethylene glycol 6000; the pellet was dissolved and refractionated by polyethylene glycol, as described for the complexes from another source [6]. The PDHC and OGDHC preparations partially purified by this method exhibited a specific activity of 1.04 and 1.34 units/mg of protein, respectively. Electrophoresis with sodium dodecyl sulfate revealed in each preparation about 15% the associated complex not preventing though from the use of these complexes for kinetic studies. PDHC from the domestic pig heart was assayed using the Sigma preparation with a specific activity of 2.3 units/mg of protein. The rates of the PDHCand OGDHC-catalyzed reactions were measured by NAD reduction at 340 nm on a spectrophotometer Beckman DU-640. The basic SHORT COMMUNICATIONS

Dissimilar properties of pyruvate kinase from rabbit and hare muscle

Some catalytic and kinetic properties of pyruvate kinase (PK, EC 2.7.1.40) isolated from rabbit and hare heart and skeletal muscle with a 9–16-fold degree of purification were studied. The initial specific activity of the enzyme in hare heart homogenates was by 66% while in hare skeletal muscle by 25% higher than in respective rabbit tissues. Temperature optimums and thermostability of PK from hare tissues were higher as compared with those in rabbits. A comparison of KM (S0.5) values shows that PK from hare skeletal muscle exhibits a highest affinity to phosphoenol pyruvate, but lowest to ADP, as compared with PK from rabbit skeletal muscle. Moreover, PK from both hare tissues exhibits a positive kinetic cooperativity (Hill coefficient > 1.35) of the phosphoenol pyruvate and ADP binding sites. In contrast to PK from rabbit tissues, the enzyme from hare heart and skeletal muscle is more sensitive to the inhibiting effect of ATP. It is suggested that in hare skeletal muscle PK is presented by its allosteric isoform which might be advantageous under extreme conditions of the hare’s habitation.