Purification and Characterization of the Glutathione-S-transferases from the Northern Quahog Mercinaria mercinaria

Glutathione-S-transferase (GST) was isolated from the northern hardshell clam Mercinaria mercinaria (quahog) using a two-step procedure involving ammonium sulfate precipitation and affinity chromatography. Kinetic analysis of the purified enzyme using 1-chloro-2,4-dinitrobenzene as substrate revealed a specific activity of 38.0 μmol min⁻¹ mg⁻¹, while V _max and K _m values were estimated as 48.0 μmol min⁻¹ mg⁻¹ and 0.24 mM, respectively. Electrophoretic analysis of GST indicated multiple forms of the dimeric enzyme in quahogs with subunit molecular masses of 22, 24, 25, and 27 kDa. Isoelectric focusing analysis resulted in pI values for three isoenzymes of 5.1, 4.9, and 4.6. The acidic pI values obtained indicated that quahog GST belongs to the π class. Inhibition of quahog GST by tetrapyrroles was similar to that of GST from oyster and rat liver. Quantitative comparison of tetrapyrrole inhibition patterns of quahog GST with those of oyster and rat liver GST indicated lower inhibition rates by three of the four tetrapyrroles tested (bilirubin, biliverdin, and chlorophillyin), suggesting that quahog GST could differ structurally or functionally from oyster and rat liver GSTs. Received March 17, 1998; accepted August 18, 1998.     

Specie's differences in glutathione-dependent enzymes in the liver and kidney of two fresh water fishes and their implications for cadmium toxicity

The protective effects of glutathione-dependent enzymes have been studied against cadmium toxicity in the liver and kidney of two fresh water fishesChanna punctatus andClarias batrachus. Specie's differences in the activity of tissue enzymes have also been studied. Cadmium treatment induced lipid peroxidation in the liver and kidney of both species, the kidney being the more susceptible. Enzymological observations revealed thatChanna punctatus is better equipped with conjugating enzymes thanClarias batrachus. Fish having higher activities of these enzymes are thus expected to withstand oxidative stress more effectively.     

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.     

Expression of CYP and GST in human normal and colon tumor tissues

We investigated the immunohistochemical staining characteristics of cytochrome P450 1A1 (CYP1A1), CYPB1, CYP2E1, and glutathione S-transferase P1 (GSTP1), GSTT1, GSTO1, GSTK1 in colon tumor and surrounding normal colon tissues. Tissues were obtained from 47 patients with colon adenocarcinoma and the staining intensity of tumor and control tissues was compared. CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTT1, GSTO1 and GSTK1 expressions in colon cancer cells were significantly greater than those in normal colon epithelial cells. No significant relation was found between the isoenzyme expressions and age, gender, smoking status, tumor grade and tumor stage. The higher expressions of CYP1A1, CYP1B1, CYP2E1, GSTP1, GSTO1, GSTT1 and GSTK1 in tumor than in normal colon tissues may be important for colon cancer progression and development.     

Antioxidant enzyme activities in subcellular fractions of larvae of the black swallowtail butterfly,Papilio polyxenes

The black swallowtail butterfly, Papilio polyxenes, larvae are specialized feeders of pro-oxidant rich plants of Apiaceae and Rutaceae. An important defense against toxic forms of oxygen species generated by ingestion of the pro-oxidants, are the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), GSH-dependent glutathione peroxidases (selenium-dependent glutathione peroxidase [GPOX] and peroxidase activity of selenium-independent glutathione-S-transferase [GT_px]), and glutathione reductase (GR). The subcellular distribution of these enzymes in black swallowtail larvae was investigated and was found to resemble the patterns described for larvae of two other lepidopteran species: the southern armyworm, Spodoptera eridania, and the cabbage looper, Trichoplusia ni. The confinement of SOD in the cytosol and mitochondria was typically eukaryotic, but the relative proportion (1:1) was markedly different from the mammalian pattern (4:1; cytosol:mitochondria). The most obvious difference between the black swallowtail and other lepidoptera as a group, and mammalian species, is in very wide intracellular distributions of CAT, GTpx, and GR in insect species. Insects possess very low levels of a GPOX-like activity which reduces both H₂O₂ and organic peroxides. Consequently, insects have elaborate activities with a wide subcellular distribution of both CAT which decomposes H₂O₂, and GTpx which decomposes organic peroxides. The reduction of peroxides is dependent on GSH, which in this process is oxidized to GSSG. GR which reduces GSSG to GSH is also of wide subcellular distribution, analogous to the distribution pattern of GTpx.