Enzymatic activity of hydrolases in the spinal cord of rat after an acute and subacute calomel introxication.

Studies have been performed on changes in the activity of hydrolytic enzymes in spinal cord following a toxic action of calomel. With the use of a gastric cannule, the rats were being administered mercurous chloride. The first group were given Hg2Cl2 in a 0-05 g. dose for four days whereas the second experimental group received a 0-012 g. for ten days. As a result of the studies, both in an acute and subacute calomel intoxication, an increase in TTP-ase and NsE activity in spinal cord as well as a decrease in ATP-ase, AChE and AcP activity were found. It was also shown that mercurous chloride intoxication exerts of influence on AIP and BuTJ activity. In the discussion an attempt was made to explain some of the observed phenomena.     

Changes of enzyme activity in the rat liver at different age in circadian cycle

Most of the biological processes in the living organisms of both animals and man are known to be of rhythmical nature. Variability of enzymatic activity in circadian cycle depends on many factors among other on age, sexual maturity, diet as well as medication. The results obtained in our studies indicate, that the activity changes of acid phosphatase and ATP-ase Mg++ dependent in the liver of all the examined age groups were of 24 hour circadian rythm. As to the acid phosphatase activity the results of this experiments showed that in circadian cycle in all examined age groups there is only one peak of elevated activity. ATP-ase Mg++ dependent showed two activity peaks appearing at the same hour both in 30 and 60 days old animals. It should be noticed that the activities of ATP-ase Mg++ dependent in 100 day old animals were two times higher than in 30 and 60 days old rats.     

Effect of parathyroid hormone and thyrocalcitonin on the cell membrane Na, K-ATP-ase of rat brain and kidney]

The influence of parathyroid hormone (PH) and thyrocalcitonine (TCT) on the enzymatic activity of ATP-ase systems of the membrane specimens of the cerebral cortex and renal cortex was investigated in experiments on rats. It was found that parathyroid hormone increased the activity of Na, K-ATP-ase and Ca-activated ATP-ase transport of the membranes in the brain and the kidneys both in vivo and in vitro. TCT caused analogous, but less expressed changes of the ATP-ase activity. Both hormones showed no influence on the Mg-ATP-ase activity of the both organs. It is supposed that the PH hormone influenced the membrane structures with the ATP-ase activity directly, while the action of TCT on them was mediated.     

Urinary enzyme excretion and renal lactate dehydrogenase isoenzyme pattern in acute HgCl2 nephropathy of rat

The activity of several enzymes with different intracellular sites was determined in urine at various times following nonfatal acute tubular necrosis induced by mercuric chloride administration. The excretion rate of all tested enzymes rose on the 1st and 2nd day; in the next observations (days 7-15) enzymatic values approached the basal values. The lactate dehydrogenase isoenzyme pattern of the renal cortical zone showed an early shift towards cathodic fractions and later (7 days) an increase of middle ones; the normal anodic zymogram recovered after a suitable time interval (30 days). The isoenzymatic changes are related both to the renal hypoxia and to the appearance of less differentiated cells. The behaviour of functional parameters (urine flow, osmolality, urea clearance, creatinine clearance) were well in agreement with the observed enzyme and renal isoenzyme changes.     

Neurotrophic control of myosin synthesis in guinea pig slow muscle]

After experimental cease of neurotrophic control of skeletal muscle by denervation no changes in myosin ATP-ase histochemistry and immunohistochemical profile in slow (m. soleus) muscle of guinea pig were found. All muscle fibers in intact muscle fibers). However after colchicine blockade of axoplasmic transport in this slow muscle some muscle fibers reacting with monoclonal antibodies against fast myosin heavy chain were found. At the same time no changes in histochemical ATP-ase profile were observed. Validity of myosin ATP-ase histochemistry for muscle fibers typing as well as possible influence of nerve activity and neurotrophic control itself were discussed.     

Partial inactivation of cytochrome c oxidase by nonpolar mercurial reagents

Purified beef heart cytochrome c oxidase is inactivated to the extent of 35 to 50% by the nonpolar mercurial reagents mercuric chloride and ethylmercuric chloride. The inactivation is complete within 5 min. In titrations of activity, the plateau level of inactivation is attained at added ethylmercuric chloride:heme a ratios of about 1:1. Up to 3 mercury atoms/heme a are bound to the oxidase, although only the first of these affects its enzymatic activity. Incubation of the ethylmercury-modified oxidase with sulfhydryl compounds reverses the inactivation, with 2,3-dimercaptopropanol being most effective of the reagents tested. Spectrophotometric and polarographic assays of enzymatic activity show that Km values for the native and the ethylmercury-modified enzymes are practically indistinguishable, and that the partial inactivation observed for the latter is reflected exclusively in a lower value of Vmax compared to that of the native enzyme. Based on these results, we propose that ethylmercuric chloride reacts with a single crucial--SH group per heme a, and that electron transfer processes in the modified product are partially inhibited.     

Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process

Rat kidney acylase I was characterised by performing site-directed mutagenesis and enzymatic analysis in the presence of various chemical inhibitors. Site-directed mutagenesis on E147 and overexpression of the protein in a bacterial system, revealed the importance of this residue in enzymatic activity, it corresponds to the putative catalytic E175 in carboxypeptidase G2 from Pseudomonas aeruginosa. The reactivity of histidine and cysteine residues of acylase I with diethylpyrocarbonate (DEPC) and mercuric chloride, respectively, showed that these two amino acids are required for the enzyme to be fully active. Interestingly, the effects of mercuric chloride on rat kidney acylase I were not as great as those on the porcine enzyme, in agreement with previously observed differences between the two enzymes. Moreover, N-[3-(2-furyl)-acryloyl-L-methionine] (FA-Met) a synthetic substrate of the porcine acylase I was found to be an inhibitor of the rat kidney enzyme. These results strongly suggest the existence of differences between the active site of rat and porcine kidney acylases I. Lastly, the rat kidney enzyme was as sensitive as its porcine counterpart to two metal chelating agents, 1,10-phenanthroline and ethylenediamine tetraacetate (EDTA).     

Na+ + K+] ATP-ase in liver and brain of obese mice

The activity of hepatic [Na+ + K+]ATP-ase showed a gene-dosage relationship in 6 week old mice. Before weaning hepatic [Na+ + K+]ATP-ase activity was normal in preobese mice but fell within 7 days of weaning to the low levels observed in older ob/ob mice. Brain [Na+ + K+]ATP-ase activity was unchanged in ob/ob mice although [3H]-ouabain binding was reduced. Arrhenius plots of [Na+ + K+]ATP-ase activity in liver and brain and of [3H]-ouabain binding to brain preparations showed breakpoints at lower temperatures in ob/ob than lean mice. These breakpoints were altered by pretreatment of tissue with deoxycholate. It is suggested that changes in membrane lipid composition might be an important factor regulating [Na+ + K+]ATP-ase in ob/ob mice.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.     

Short Communication: Urinary excretion of glutamine transaminase K as an early index of mercuric chloride-induced nephrotoxicity

The possibili that urinary glutamine transaminase K activity might be a marker of a proximal tubule segment-specific response to mercuric chloride was investigated in male rats after a single i.p. injection in time-course and dose-response experiments. Urinary total proteins and angiotensin converting enzyme activity were determined simultaneously. Urinary indices showed an early increase (within 5 h of treatment) of total proteins and angiotensin converting enzyme, whereas glubmine transaminase K increased 10 h after treatment. The peak of all these indices was observed 24 h after mercuric chloride injection. The lowest dose that induced a significant increase in proteins and enzymes was 0.25 mg kg^-1; in addition, a dose-response effect was observed. Glutamine transaminase K appeared to be an early and sensitive index of response of mercuric chloride effects, similar to total proteins and angiotensin converting enzyme. It is suggested that this enzyme is mainly localized in the 'pars recta' of the proximal tubule. Therefore glutamine transaminase K might be a segment-specific marker for the detection of damage localized in this portion of the proximal tubule.     

Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Uptake and subcellular cleavage of organomercury compounds by rat liver and kidney

The uptake and transformation of phenylmercuric acetate (PMA), ethylmercury chloride (EMC), and methylmercury chloride (MMC) by rat liver, kidney, and brain slices were investigated. A slightly greater enzymatic activity for the cleavage of PMA and EMC was observed in the kidney than in the liver; brain tissue had the least activity.The average rates of C-Hg cleavage were 2.4 to 3.0 nmole g^?1 h^?1 for PMA and 0.9 to 1.0 nmole g^?1 h^?1 for EMC. No cleavage of C-Hg bond from MMC was observed by the kidney and liver.The cleavage of PMA was an enzymatic process and the enzyme was located in the nuclear fraction of both liver and kidney. The apparent Michaelis constant and the maximal velocity value for PMA conversion by rat liver slices were 625 μM and 19.2 nmole per g tissue per 2 h, respectively.It is postulated that the PMA molecule is first split at the C-Hg bond to yield benzene and mercuric ion.The cleavage of the EMC molecule appears to be more complex.     

The Inflammatory Potential of Dietary Manganese in a Cohort of Elderly Men

In the current study, 48 male rats were classified into four groups (12 rats/group): 1—control group received 1 ml distilled water, 2—origanum oil group treated daily with oral dose of origanum oil (5 mg/kg) for 30 and 60 days, 3—mercuric chloride group treated daily with oral dose of mercuric chloride (4 mg/kg) for 30 and 60 days, and 4—origanum oil + mercuric chloride group treated with both origanum oil and mercuric chloride (5 and 4 mg/kg, respectively) for 30 and 60 days. All treatments were carried out by stomach tube. The results showed that administration of mercuric chloride induced significant increase in thiobarbituric acid reactive substance (TBARS) and decrease in glutathione (GSH), catalase (CAT), and super oxide dismutase (SOD) in testis and spleen tissues. The data also showed significant increase in tumor necrossis factor-α (TNF-α), 8-hydroxy deoxyguanosine (8-OHDG), acid phosphatase (ACP), urea, and creatinine. Furthermore, significant decreases in serum zinc (Zn), copper (Cu), magnesium (Mg), iron (Fe), and testosterone in mercuric chloride group were recorded. The histological examination of testis and spleen tissues showed some degenerative changes while significant improvement in the antioxidant levels, biochemical, trace elements, and histological changes were observed in mercuric chloride group treated with origanum oil. It could be concluded that origanum oil through its antioxidant potential may possess health promoting properties and could protect cells from oxidative damage induced by mercuric chloride.     

Ultracytochemical study of ATP-ase activity in mucosal parietal cells and in the cells of human adenocarcinoma of the stomach]

Ultracytochemical investigation of ATP-ase activity was carried out in parietal cells of the mucosa and in cancer cells of human stomach carcinoma possessing a similar ultrastructure. In parietal cells the reaction product of ATP-ase was observed on the membranes of microvilli of intracellular canaliculi, on the membranes delineating the lateral intercellular space, on the basal plasmolemma and in the nucleoli. The reaction product was absent on the membranes of tubuvesicles and on the apical surface of the plasmalemma. In cancer cells the reaction product was found on the membranes of the microvilli of the intracellular canaliculi, basal plasmolemma and in the nucleoli. Comparative examination of ATP-ase activity in these cells implies that at least the part of the mechanism of hydrochloric acid secretion which is involved in the transfer of H+ and Cl- is retained in cancer cells. A steady decrease in hydrochlorid acid secretion observed in the stomach mucosa in cancer as well as in the tumour itself seems to be associated with other mechanisms.     

Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Uptake of mercury and mercury-amino acid complexes by rat renal cortex slices.

This study was undertaken in order to assess the effects of metabolism and complexations with amino acids on the renal uptake of mercury using rat renal cortex slices as the experimental system. Mercury levels attained in the slices after 60 min of incubation were 50% higher with mercuric cysteine than with mercuric chloride. This enhancement of uptake with mercuric cysteine was reduced in the presence of a tenfold molar excess of histidine or lysine, but not by serine. Excess cysteine markedly increased mercury uptake. Incubation at 25 degrees significantly reduced uptake of mercuric cysteine, but not mercuric chloride. Anaerobic conditions and incubation in the presence of DNP each reduced mercuric cysteine uptake to the control level of mercuric chloride without affecting uptake of mercuric chloride. The differential aspects of metabolism on the uptake of mercuric cysteine and mercuric chloride and the competitive effects obtained with amino acids known to compete with cysteine in renal reabsorption support the hypothesis that a portion of the renal uptake of mercury operates through amino acid transport mechanisms acting on mercury-amino acid complexes.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Effect of vasopressin on the ATP-ase activity of microsomal fractions of rabbit heart and liver]

It was shown that intravenous injection of vasopressin in a dose of 5 pressor units per 1 kg of body weight led to changes in the ATP-ase activity of the heart and liver microsomes in one hour. These changes coursed in a different direction, i.e. ATP-ase activity of the heart microsomes increased, and ATP-ase activity of the liver microsomes decreased.     

Changes in the activity of hydrolytic enzymes in the brain of rats intoxicated by ethyl-mercury-p-toluene-sulfanilide.

A histochemical study concerning the activity of phosphatases and esterases of the brain has been undertaken in rats experimentally intoxicated by the fungicide ethyl-mercury-p-toluenesulfanilide (EMTS). The results have shown that compared with other mercury compounds, both organic and inorganic ones, such as corrosive sublimate and calomel, EMTS proved to be a less induced of alterations in the activity of cerebral hydrolases. The brains of animals intoxicated by EMTS revealed a notable decrease of ATP-ase and acid phosphatase activity as well as a moderate drop of AChE activity. Instead, the neuronal TPPase activity was distinctly elevated. Degenerative changes of neurons were observed in various regions of the experimental brains, the pyramidal cells of the Ammon's horn being affected most severely.