Aspects of the hemes and modulation of hydrogen donors in catalases from bovine liver, yeast, and escherichia coli

Catalase is the enzyme which decomposes hydrogen peroxide to water and oxygen. Escherichia coli contains two catalases. Hydroperoxidase I (HPI) is a bifunctional catalase-peroxidase. Hydroperoxidase II (HPII) is only catalytically active toward H202. Expression of the genes encoding these proteins is controlled by different regimes. HPJI is thought to be a hexamer, having one heme d cis group per enzymatic subunit. HPII wild type protein and heme containing mutant proteins were obtained from the laboratory of P. Loewen (Univ. of Manitoba). Mutants constructed by oligonucleotidedirected mutagenesis were targeted for replacement of either the His128 residue or the Asn201 residue in the vicinity of the HPII heme crevice. His128 is the residue thought to be analogous to the His74 distal axial ligand of the heme in the bovine liver enzyme, and Asn201 is believed to be a residue critical to the function of the enzyme because of its role in orienting and interacting with the substrate molecule. Investigation of the nature of the hemes via absorption spectroscopy of the unmodified catalase proteins and their derived pyridine hemochromes showed that while the bovine and Saccharomyces cerevisiae catalase enzymes are protoheme-containing, the HPII wild type protein contains heme d, and the mutant proteins contain either solely protoheme, or heme d-protoheme mixtures. Cyanide binding studies supported this, as ligand binding was monophasic for the bovine, Saccharomyces cerevisiae, and wild type HPII enzymes, but biphasic for several of the HPII mutant proteins. Several mammalian catalases, and at least two prokaryotic catalases, are known to be NADPH binding. The function of this cofactor appears to be the prevention of inactivation of the enzyme, which occurs via formation of the inactive secondary catalase peroxide compound (compound II). No physiologically plausible scheme has yet been proposed for the NADPH mediation of catalase activity. This study has shown, via fluorescence and affinity chromatography techniques, that NADPH binds to the T (Typical) and A (Atypical) catalases of Saccharomyces cerevisiae, and that wild type HPII apparently does not bind NADPH. This study has also shown that NADPH is unlike any other hydrogen donor to catalase, and addresses its features as a unique donor by proposing a mechanism whereby NADPH is oxidized and catalase is protected from inactivation via the formation of protein radical species. Migration of this radical to a position close to the NADPH is also proposed as an adjunct hypothesis, based on similar electron migrations that are known to occur within metmyoglobin and cytochrome c peroxidase when reacted with H202. Validation of these hypotheses may be obtained in appropriate future experiments.

An investigation into differences between indoleacetic acid and fusicoccin in their influence on RNA synthesis, protein synthesis and growth in Avena coleoptile tissue

Growth stimulation of Avena coleoptile tissue by indoleacetic acid (IAA) and fusicoccin (FC) was compared by measuring both their influence on RNA and protein synthesis during IAA or FC stimulated growth. FC stimulated growth more than IAA during the initial four hour exposure, after which the growth rate gradually declined to the control rate. FC, but not IAA, increased the uptake of 3H-Ieucine into tissue and the specific radioactivity of extracted protein. Cycloheximide inhibited the incorporation of 3H-Ieucine into protein by approximately 60% to 70% in all cases. In the presence of cycloheximide 3H-radioactivity accumulated in FC-treated tissue, whereas IAA did not seem to influence 3H-accumulation. These results suggest that FC stimulated leucine uptake into the tissue and that increased specific activity of coleoptile protein is due to increased leucine uptake, not an increased rate of protein synthesis. There was no measurable influence of IAA and/or FC on RNA and protein synthesis during the initial hours of a growth stimulation. Inhibitors of RNA and protein synthesis, actinomycin D and cycloheximide, respectively, severely inhibited IAA enhanced growth but only partially inhibited FC stimulated growth. The data are consistent with suggestions that a rapidly turning over protein participates in IAA stimulated growth, and that a continual synthesis of RNA and proteins is an absolute requirement for a long term growth response to IAA. On the contrary, FC-stimulated growth exhibited less dependency on the transcription and translation processes. The data are consistent with proposals suggesting different sites of action for FC and IAA stimulated growth. l?hen compared to CO2-free air, CO2 at 300 ppm had no significant influence on coleoptile growth and protein synthesis in the presence or absence of lAA or FC. Also, I mM malate, pH 6.0 did not influence growth of coleoptiles in the presence or absence of lAA. This result was obtained despite reports indicating that 300 ppm CO2 or I mM malate stimulates growth and protein synthesis. This lack of difference between CO2-treated and untreated tissue could indicate either that the interstitial space CO2 concentration is not actually different in the two treatments due to significant endogenous respiratory CO2 or else the data would suggest a very loose coupling between dark CO2 fixation and growth. IAA stimulated the in vivo fixation of 14c-bicarbonate (NaHI4c03) by about 25% and the addition of cycloheximide caused an inhibition of bicarbonate fixation within 30 min. Cycloheximide has also been reported to inhibit IAA-stimulated H+ excretion. These data are consistent with the acid growth theory and suggest that lAA stimulated growth involves dark CO2 fixation. The roles of dark CO2 fixation in lAA-stimulated growth are discussed.

Human skeletal muscle pyruvate dehydrogenase phosphatase activity and expression : the effect of aerobic capacity

Activation of pyruvate dehydrogenase (PDH), which converts pyruvate into acetyl-CoA, is accomplished by a pair of specific phosphatases (PDP 1 & 2). A cross-sectional study investigating the effect of aerobic capacity on PDP activity and expression found that: 1) PDP activity and PDP! protein expression were positively correlated with most aerobic capacity measures in males (n=lS), but not females (n=12); 2) only males showed a positive correlation between PDP activity and PDPl protein expression (r=0.47; p=O.05), indicating that the increase in PDP activity in males is largely explained by increased PDPl protein expression, but that females rely on another level for PDP activity regulation; and 3) PDP} and Ela protein expression increase in unison when expressed relative to the E2 core. These data suggest that with increased aerobic capacity there is an increased capacity for carbohydrate oxidation through PDH, via El a, and an increased ability to activate PDH, via PDP, when exercising maximally.

Carbohydrate refeeding rapidly reverses the adaptive upregulation of human skeletal muscle pyruvate dehydrogenase kinase following a high fat diet

The time course for the reversal of the adaptive increase in pyruvate dehydrogenase kinase (PDK) activity following a 6d high fat diet (HP: 4.2 ± 0.2 % carbohydrate; 75.6 ± 0.4 % fat; 19.5 ± 0.8 % protein) was investigated in human skeletal muscle (vastus lateralis). HF feeding increased PDK activity by 44% (from 0.081 ± 0.025 min"' to 0.247 ± 0.025 mm\p < 0.05). Following carbohydrate re-feeding, (88% carbohydrate; 5% fat; 7% protein), PDK activity had returned to baseline (0.111 ± 0.014 min"') within 3h of re-feeding. The active fraction of pyruvate dehydrognease (PDHa) was depressed following 6d of the HF diet (from 0.89 ± 0.21 mmol/min/kg WW to 0.32 ± 0.05 mmol/min/kg ww,p <0.05) and increased to pre-HF levels by 45 min of post re-feeding (0.74 ±0.19 mmol/min/kg ww) and remained elevated for 3h. Western blotting analysis of the PDK isoforms, PDK4 and PDK2, revealed a 31% increase in PDK4 protein content following the HF diet, with no change in PDK2 protein. This adaptive increase in PDK4 protein content was reversed with carbohydrate re-feeding. It was concluded that the adaptive up-regulation in PDK activity and PDK4 protein content was fiilly reversed by 3h following carbohydrate re-feeding.