Decrease of rat liver cysteine dioxygenase (cysteine oxidase) activity mediated by glucagon.

The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.     

Two components of cysteine oxidase in rat liver

Purified cysteine oxidase in rat liver is composed of two distinct proteins. These proteins are able to be fractionated by DEAE-cellulose column chromatography. It appears that one of them is a catalytic protein named protein-B having tightly bound iron as a prosthetic group, while the other is either a modifier or activating protein named protein-A. Protein-B is found to exist in both an active and an inactive form. Inactive protein-B is activated by incubation with substrate cysteine under anaerobic condition. Activated protein-B alone exhibited an extremely low catalytic activity but in the presence of protein-A remarkable increase in activity was observed.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation

Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.     

The influence of respiratory state on monoamine oxidase activity in rat liver mitochondria.

Changes in the respiratory state of rat liver mitochondria caused significant changes (up to 10-fold) in the rates of oxidative deamination of tyramine, indicating interactions between the inner coupling membrane and the monoamine oxidase sites in the outer membrane, and suggesting the possibility that monoamine oxidase is regulated by the thermodynamic state of the mitochondria.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

Morphine diesters. II. Blood metabolism and analgesic activity in the rat

The kinetics of hydrolysis of dipropanoylmorphine (DPM) and dibutanoylmorphine (DBM) in human blood fractions and for diacetylmorphine (DAM) and DBM in rat blood fractions were investigated. In each case the hydrolysis of morphine diesters terminated with the production of the corresponding 6-monoester derivative. Generally, decreases in Km and Vmax were observed for the plasma, red blood cell (RBC) cytosol, and RBC membrane esterases responsible for morphine diester hydrolysis as the alkyl chain length of the ester moiety increased. This resulted in an overall decrease in the rate of hydrolysis of morphine diesters by human or rat blood with longer chain homologs of DAM. The analgesic potency and duration of morphine, DAM, and DBM were assessed at various i.v. dosages in the rat by means of the tail-flick latency test. A comparison of equianalgesic doses of morphine, DAM, and DBM indicated that DAM and DBM were 11.5 and 6 times as potent and 0.8 and 1.2 times as long acting, respectively, as morphine.     

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.     

Subcellular distribution of cysteine oxidase activity in ox retina.

Cysteine oxidase activity has been determined in the primary and secondary subfractions of ox retina. About 30% of enzyme activity is found in the soluble fraction while about 70% is associated with particulate components.In the secondary subcellular fractions about 36% of enzyme activity, recovered from crude mitochondria, is present in the synaptosomal fraction.Enzymic activity is stimulated by Fe⁺⁺ and NAD⁺. The reason and significance of the cysteine oxidase activity in synaptosomal fraction are briefly discussed in relation with taurine function in retina.     

In vitro effects of corticosteroids, DDT, and 4, 9-dichlorodibenzodioxin on rat liver xanthine oxidase activity. Interactions between xanthine oxidase and cytochrome P450 in rat liver in vivo

It was established that deoxycorticosterone, cortisol, dexamethasone, DDT, and 4,9-dichlorodibenzodioxin inhibit in vitro binding of xanthine to highly purified rat liver xanthine oxidase. They are suggested to be allosteric inhibitors. The corresponding inhibition and binding constants were estimated. Also established was that cortisol and DDT, in dose 20 mg per kg of weight, inhibit xanthine oxidase and increase microsomal cytochrome P450 level in rat liver. There is an inverse relationship between xanthine oxidase activity and cytochrome P450 level in rat liver.