Proline oxidase in cultured mammalian cells

We sought a cultured cell line with Proline Oxidase activity to study the regulation and physiologic role of the enzyme in mammalian tissues. Among the cell lines tested, only LLC-RK1 cells, derived from rabbit kidney, had significant Proline Oxidase activity; the Km for proline of the enzyme from these cells was similar to that for the liver enzyme. LLC cells, Proline Oxidase positive, were able to convert proline to CO2. In contrast, CHL cells, Proline Oxidase negative, did not have this capability. The presence of Proline Oxidase in LLC cells and the absence of the enzyme in fibroblasts suggest that Proline Oxidase may serve as a marker enzyme for distinguishing parenchymal kidney cells from fibroblasts in culture. Cells transformed by SV40 virus and cells transformed by methylcholanthrene had activities higher that the parent cell line, but this effect of transformation could not be generalized to all transformed cells. Finally, L-hydroxy proline at 100-fold greater concentration than substrate L-proline failed to decrease proline oxidation. This finding suggests distinct degradative enzymes for these two amino acids.     

Tyramine oxidase activity in needle biopsy of normal livers and diseased livers.

Tyramine oxidase and UDP-glucuronyl transferase activities were determined in 52 diseased livers obtained by needle biopsy. 14 liver specimens were also subjected to acetyl CoA carboxylase determination. Tyramine oxidase level was elevated in livers with nonalcoholic fatty change or toxic hepatitis, and reduced in livers with fibrosis or chronic alcoholic injury. UDP-glucuronyl transferase activity was reduced in livers with severe parenchymal damage or hyperbilirubinemia. Acetyl CoA carboxylase activity decreased markedly in an active alcoholic cirrhotic liver, and was elevated in alcoholic fatty livers as well as in a liver with acute venous stasis.     

Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam

The two species common of liver fluke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these species, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver flukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoonotic disease. Specimens examined came from domestic stock (cattle and buffalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia.     

Comparative studies on the molecular weight of glutathione S-transferases from mammalian livers and an insect.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.     

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.     

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.     

Activities of glucokinase and hexokinase in mammalian and avian livers.

A radiochemical assay for glucokinase activity was developed for use in high-speed supernatants of liver. The maximum activities of glucokinase ranged from 0.4 to 3.8 mumol/min per g fresh wt. at 30 degrees C in some avian and mammalian livers, including pigeon, guinea pig and man, in which previous reports indicated zero activities. The reported maximum rates of hepatic glycogen synthesis in livers of rat and man in vivo are similar to the calculated glucokinase activities at 10mM-glucose; therefore glucokinase activity should not limit glycogen synthesis from glucose.     

Schistosoma mansoni: partial purification and properties of ornithine-delta-transaminase.

Ornithine-δ-transaminase (OTA) (EC 2.6.1.13) was isolated from Schistosoma mansoni and purified more than 16-fold. Treatment of the worm homogenate with 0.4% deoxycholate (DOC) in the presence of 0.8 M KC1 and 0.15 M NaCl at pH 8.3 resulted in solubilization of 85% of the enzyme. Sonication and high-speed centrifugation were unnecessary. The solubilization procedure and the subsequent purification steps required the presence of the coenzyme pyridoxal phosphate. The optimal pH for OTA was 8.5 and the optimal incubation temperature was 55 C. Michaelis-Menten constants (K_m) for ornithine and α-ketoglutarate were 1.53 mM and 2.07 mM, respectively, in enzyme preparations with a specific activity of 22–29 μmoles/hr/mg protein. The enzyme showed a high affinity for α-ketoglutarate but considerably less affinity for oxaloacetate and pyruvate. High concentrations of α-ketoglutarate and ornithine inhibited the OTA activity. Similarly inhibitory were the structurally related amino acids isoleucine and serine and also oxaloacetate. The K_m for α-ketoglutarate in the presence of oxaloacetate was 1.3 mM and the V_max was 8.38 μmoles/hr/mg protein.     

Proline metabolism in pollen: degradation of proline during germination and early tube growth

Proline degradation inPetunia pollen germinated in vitro was studied in cultures supplemented with [¹⁴C]proline labeled at different positions. Despite its abundance in the cell, this amino acid is only a minor substrate for respiration. Proline is partially converted via the citrate cycle into metabolites which can be traced in the ethanol-insoluble fraction of the cellular constituents. The fact that this conversion is much more extensive than the use of proline in respiration indicates that, in germinating pollen, the citrate cycle serves mainly for synthetic purposes. There are no indications that proline carbon is used for feeding of other amino-acid pools used for protein synthesis.     

Liver glycogen metabolism in endotoxin shock. I. Endotoxin administration decreases glycogen synthase activities in dog livers

The effects of E. coli endotoxin administration on hepatic glycogen content and glycogen synthase activities in dogs were studied. Liver glycogen content was decreased by 80% 2 hr after endotoxin injection. When enzyme preparations were preincubated at 25 degrees C for 3 hr prior to their assays, 75% of total glycogen synthase was in I form in control dogs. Under such conditions, endotoxin administration decreased the percentage I activity from 75 to 37%; decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 62.2 and 35.3%, respectively; decreased the Vmax and Km for UDP-glucose for glycogen synthase I by 75.6 and 15.6%, respectively; increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 126% at high (10 mM) and by 18-fold at low (1 mM) UDP-glucose concentration; increased the percentage D activity from 24 to 72%; decreased the I50 for ATP for the inhibition of total glycogen synthase by 49.7%; decreased the I50 for ATP for the inhibition of glycogen synthase I by 26.4%; and decreased the percentage I activity from 78 to 33% at ATP concentrations below 6 mM. When enzyme preparations were not preincubated prior to their assays, 90% of total glycogen synthase was in D form in control dogs. Under such conditions, endotoxin administration decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 47.1 and 33.3%, respectively, and increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 24.2% at high (10 mM) and by 106% at low (1 mM) UDP-glucose concentration. From these results, it is clear that endotoxin administration greatly impaired hepatic glycogenesis by decreasing the activity of glycogen synthase; this impairment is at least in part responsible for the depletion of liver glycogen content in endotoxin shock. Kinetic analyses revealed that the decrease in the activity of glycogen synthase in endotoxic shock is a result of a decrease in the interconversion of this enzyme from inactive to active form and an increase in the interconversion from active to inactive form.     

On the compartmentalization of catalase,fatty acyl-CoA oxidase and urate oxidase in mammalian livers,and the influence of clofibrate treatment on this microlocalization

The compartmentalization of catalase, fatty acyl-CoA oxidase and urate oxidase was examined in the livers of mice, rats and guinea pigs, using the technique of digitonin extraction in order to avoid the trauma associated with centrifugation procedures. The results are interpreted as indicating that an appreciable proportion of catalase activity occurs in the cytoplasmic compartment of these cells. Following treatment of the animals with clofibrate, the specific activity in both peroxisomal and cytoplasmic compartments was increased, with a higher proportion of cytoplasmic catalase being evident in mice. The results for catalase were compared with those for fatty acyl-CoA oxidase and urate oxidase both of which were indicated as showing a closer association with the peroxisomal compartment than was the case for catalase. These data have been discussed in relation to their significance on present understanding of peroxisomal structure and function.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

Schistosoma mansoni: higher free proline levels in the livers of infected mice

The concentration of L-hydroxyproline in the liver of ICR female mice increased rapidly during the 8th to 11th weeks of Schistosoma mansoni infection. Free L-proline concentration began to increase about the 7th week and reached its maximum at the 8th to 9th weeks of the infection, when the granulomatous response to the schistosome eggs in the liver was most prominent, as indicated by the increase in liver wet weight and its deoxyribonucleic acid concentration. A significant increment in the total activity of ornithine-delta-transaminase (EC 2.6.1.13) and the decrease in the specific activity of proline oxidase (EC 1.4.3.2) became detectable in the liver homogenate of infected mice on the 8th week. However, changes in these enzymatic activities were not parallel to that of the hepatic free L-proline content. Intraperitoneal administration of S. mansoni egg granulomas or 15,000g x 30 min supernatant fluid of their extracts into uninfected, normal mice significantly increased the hepatic free L-proline content without any appreciable effect on the enzymatic activities of proline oxidase and ornithine-delta-transaminase. These findings suggest that S. mansoni egg granulomas contain a factor(s) which may be responsible for the elevation of free L-proline content in the fibrotic liver caused by experimental schistosomiasis mansoni.