Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro

A single intraperitoneal injection of dimethyl sulfoxide (275 mg/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2-5-fold. The enzyme activity remained at this level for as long as 5 days post-injection. There was however only 10.5% increase in the content of cytochromes a and a3 (as determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2-3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Dehydroepiandrosterone (DHEA) facilitates liver regeneration after partial hepatectomy in rats

In this study we investigated whether or not liver regeneration is facilitated by dehydroepiandrosterone (DHEA) after partial (70%) hepatectomy in rats. Treatment with DHEA (300 mg/kg body weight) did not cause any significant increase in the expression ratio of proliferating cell nuclear antigen (PCNA) in sham-operated controls; however, in partially hepatectomized rats it caused a significant increase in the ratio in hepatocytes 24 and 36 hr after hepatectomy. In partially hepatectomized rats, DHEA treatment significantly accelerated the restoration of liver 48, 60, and 72 hr after partial hepatectomy. The restoration rate in DHEA-treated hepatectomized rats at 72 hr was 1.3-fold greater than in partially hepatectomized controls. Treatment with androstenedione (300 mg/kg body weight), the first metabolite of DHEA, did not cause any significant increase in the expression of PCNA in either sham-operated controls or partially hepatectomized rats. These results indicate that DHEA itself promotes the liver regenerative process after partial hepatectomy in rats.     

Thermosensitive respiratory deficiency in yeast associated with specific effects on particulate cytochrome oxidase.

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37 degrees C, has been selected; at 25 degrees C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a,a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation.     

Effect of chemical carcinogens and partial hepatectomy on "in vivo" (35S)methionine interaction with rat liver tRNA

The effect of carcinogens given by a single or multiple injections on the extent of (35S)methionine interaction with hepatic tRNA was studied in normal and partially hepatectomized rats. Either partial hepatectomy or administration of ethionine (100 or 330 mg/kg body weight) and dimethylnitrosamine (120 mg/kg body weight) by multiple i.p. injections inhibited the (35S)methionine-tRNA interaction, while administration of hepatocarcinogenic chemicals plus PH resulted rather in a stimulation. Methylnitrosourea enhanced the extent of interaction when administered in a single dose (100 mg per kg body weight) 18 h after partial hepatectomy.     

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

Effect of an inhibiting factor isolated from rat liver on DNA polymerases in regenerating rat liver.

We partially purified an inhibitory factor (LIFE), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G1--S transition, and reduced in vivo [3H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented alpha DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on alpha DNA polymerase was detected. LIF did not affect beta DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.     

Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

EFFECT OF AN INHIBITING FACTOR ISOLATED FROM RAT LIVER ON DNA POLYMERASES IN REGENERATING RAT LIVER

We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G₁—S transition, and reduced in vivo [³H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented α DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on α DNA polymerase was detected. LIF did not affect β DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

NADPH oxidase of guinea-pig macrophages catalyses the reduction of ubiquinone-1 under anaerobic conditions.

The stimulation-specific NADPH-dependent reduction of ubiquinone-1 (Q-1) in guinea-pig macrophages was studied. The activity was due neither to any modified product of the phagocytosis-specific NADPH oxidase nor to non-specific diaphorases of the cells, since the activity was measured in sonicated or detergent-disrupted cells by subtracting the activity in the resting cells from that in cells activated by phorbol 12-myristate 13-acetate. The activity was not mediated by superoxide anions, since strict anaerobic conditions were employed. The anaerobic reduction of Q-1 was NADPH-specific, like superoxide formation under aerobic conditions, and its maximal velocity was also essentially the same as that of superoxide formation. The oxidase does not directly reduce Q-1 under aerobic conditions [Nakamura, Murakami, Umei & Minakami (1985) FEBS Lett. 186, 215-218], and the electron transfer from NADPH to cytochrome c by the oxidase under aerobic conditions was not enhanced by the addition of Q-1. The observations indicate that the phagocytosis-specific NADPH oxidase reduces Q-1 and that oxygen competes with the reduction of Q-1. Q-1 seems to accept electrons not from the intermediary electron carriers of the oxidase but from the terminal oxygen-reducing site of the enzyme.     

Enzymatic oxidation of bilirubin by intestinal mucosa

Bilirubin oxidase, an aerobic enzyme which degrades bilirubin 'in vitro' to colourless diazo-negative compounds, including propentdyopents and trace amounts of biliverdin, has been demonstrated in homogenates of rat intestine, kidney and liver. The enzyme in the intestinal mucosa has been partially characterised and appears to be mitochondrial in origin; maximal activity was detected in the jejunum. Intestinal bilirubin oxidase has a mean activity of 0.51 +/- 0.03 (S.D.) nmol bilirubin degraded/min per mg protein. Similar bilirubin oxidase activities were found in the tissue of Sprague-Dawley and Gunn rats. The role of the enzyme 'in vivo' remains to be determined.     

Cadmium sensitivity differences between liver microsomal drug metabolizing enzyme systems of guinea-pig and rat

1. Male guinea-pigs (400-500 g) and rats (225-275 g) were given a single dose of cadmium chloride (CdCl2) (2 mg Cd2+/kg i.p.) and 72 hr later the liver microsomal drug metabolizing enzyme activities and Cd levels of tissues and microsomes were determined. 2. No significant differences were noted between Cd treated and control animal tissue weights of microsomal protein contents in either guinea-pigs or rats. 3. Cd treatment exhibited significant inhibition of the activities of aniline 4-hydroxylase and ethylmorphine N-demethylase and on the levels of cytochrome P-450 and cytochrome b5 of liver of both species but the degree of inhibition were not the same in the species; they were 23, 34, 16 and 10% in guinea-pigs and 58, 57, 25 and 13% in rats, respectively. 4. No activity changes were observed in liver NADPH-cytochrome c reductase of the species by Cd treatment. 5. The duration of hexobarbital sleeping time was significantly prolonged in both species. However, the prolongation was 1.6 fold in guinea-pigs but 3.4 fold in rats. 6. No significant differences were found between either tissue or microsomal Cd levels of guinea-pigs and rats.     

Induction of pinocytosis in rat hepatocytes by partial hepatectomy

Rat hepatocytes, normally not highly pinocytic cells, becomes so after partial hepatectomy when about two-thirds of the liver is removed. Droplets, up to 20 mum in diameter, develop, initially by addition to smaller pinocytic structures and later by fusion with lysosomes. The droplets contain a material with an electron microscope periodicity characteristic of fibrin; they are periodic acid Schiff-positive as is plasma. It is therefore reasonable to consider plasma glycoproteins to be major components of the droplets. The droplets are at all times membrane delimited, an observation possible only after perfusion fixation. The droplets are positive for three lysosomal hydrolases identified cytochemically: acid phosphatase, N-acetyl-beta-glucosaminidase, and beta-glucuronidase. From light and electron microscopy it is evident that these activities are acquired by fusion with lysosomes, mostly autophagic vacuoles and residual bodies both of which become very numerous after partial hepatectomy. Pinocytic structures are seen relatively infrequently in the hepatocytes of normal rats but a great many are present after partial hepatectomy. They are most easily observed if horseradish peroxidase (HRP) is intravenously injected before sacrifice and sections are incubated for HRP cytochemistry. The low dose of HRP employed (10 mg/100 g body weight) does not induce pinocytosis in controls, either untreated rats or rats subjected to laparotomy, including palpation of the liver. However, in partially hepatectomized rats even a much smaller dose of intravenous HRP (3.3 mg/100 g) visualizes the pinocytic structures in hepatocytes (coated vesicles, channels, cuplike bodies, and droplets). Kupffer cells pinocytose much HRP in both control and partially hepatectomized rats.     

Metabolic control mechanisms in mammalian systems. Hormonal regulation of phosphofructokinase in the rat prostate and seminal vesicles

1. The hormonal regulation of phosphofructokinase was investigated in the accessory reproductive organs of the orchidectomized rat. 2. Phosphofructokinase activities declined to 51% and 47% in the prostate and 9% and 6% of the normal values in seminal vesicles 4 and 8 weeks after castration respectively. Administration of testosterone (100mug./100g. body wt.) for 3 days reversed substantially the effects of orchidectomy, and phosphofructokinase activity increased to 173% in the prostate and 536% in seminal vesicles as compared with the values of castrated controls. 3. Time-course studies demonstrated that after a single injection of testosterone (5mg./100g. body wt.) phosphofructokinase activity was maximally elevated to 236% in the prostate and 342% in seminal vesicles at 24hr. 4. Dose-response studies revealed that 2.5mg. of testosterone propionate/100g. body wt. was the minimal amount necessary to induce significant increases in enzyme activity in both accessory sex organs; maximal increases were obtained with a dose of 5mg./100g. body wt. 5. The observed enzyme increases induced by testosterone were inhibited by the simultaneous administration of oestradiol-17beta, and phosphofructokinase activity in this group of rats remained at 97% in the prostate and 137% of the control values in seminal vesicles. Oestradiol-17beta by itself failed to produce any significant effect on enzyme activity in either of these secondary sexual tissues. 6. The nature of the testosterone-induced increases in phosphofructokinase activity was studied by using a variety of inhibitors of RNA and protein synthesis. Cycloheximide, 5-fluorouracil and ethionine largely blocked the androgen-stimulated rise in enzyme activity observed 24hr. after steroid injection. The inhibitory effect of ethionine was completely reversed by the simultaneous administration of methionine. 7. Actinomycin, which is known to inhibit the synthesis of messenger RNA as well as the synthesis of other cellular RNA fractions, when given simultaneously with the hormone, also inhibited the testosterone-induced increases in prostatic and seminal-vesicular phosphofructokinase. However, when the antibiotic was given 6 or 12hr. after injection of the steroid, practically no inhibition of phosphofructokinase induction was obtained. This indicates that, once the enzyme-forming machinery is turned on and allowed to operate for a few hours, actinomycin is incapable of reversing the hormone-induced enzyme responses. 8. The results presented suggest that new RNA and protein synthesis may be involved in the observed androgen-induced increases in phosphofructokinase activity in the prostate and seminal vesicles of the orchidectomized rat.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

Microbial water relations. Effects of solute concentration on the respiratory activity of sugar-tolerant and non-tolerant yeasts.

The respiratory activity of the sugar-tolerant (osmophilic) yeast, Saccharomyces rouxii, and the non-tolerant species, Sacchromyces cerevisiae, were compared after growth in a complex basal medium, the medium supplemented with polyethylene glycol (mol. wt 200) to give a water activity of 0-95, and the medium supplemented with glucose (24 and 36%, w/v). The properties compared were Qo2 (glucose), NADH oxidase activity of isolated mitochondrial fractions, and cytochrome content. When grown in the basal medium S. cerevisiae was somewhat more active than S. rouxii by all criteria. Growth in the media supplemented were high glucose concentrations produced catabolite repression of respiration in S. cerevisiae but not in S. rouxii. The implications of this difference for polyol biosynthesis and the water relations of the sugar-tolerant species are discussed.     

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.     

Variations in epidermal cytochrome oxidase activity after local irradiation

Summary Cytochrome oxidase activity was evaluated histochemically as an index of mitochondrial damage after local irradiation with X-rays. It was determined by microphotometry on the tail skin of newly born Wistar rats four days after irradiation with doses ranging from 2 to 16 krad.The enzyme activity of the whole epidermis increased after irradiation, the increases being related to the increase in thickness of the epithelium which was observed as a response to irradiation injury. Within the dose range tested, the enzyme concentration (expressed per unit volume of tissue) decreased in relation to the dose applied.At the electron microscopy level, the cytochemical demonstration of cytochrome oxidase revealed an irregular reaction over the cristae, intramitochondrial vacuolization and partial homogenization of the matrix. Positive membrane fragments were seen around lipid droplets. This reaction confirms the mitochondrial origin of these previously observed radiation-induced vacuoles.