Kinds Of Isozymes Research Articles

Induction of Laccase Production and Analysis of Laccase Isozymes in Trichophyton rubrum LKY-7

Trichophyton rubrum LKY-7 is a wood-degrading fungus, which has been isolated from hardwood chip piles. T. rubrum LKY-7 showed better growth in glucose-peptone medium (GPM) than in basal medium (BM) with rotary shaking, and mycelial growth was facilitated by the presence of wood powder. Aeration significantly increased the production of laccase in GPM (6 to 17 U/mL), and the addition of wood powder to the medium greatly increased it to 78.5 U/mL. T. rubrum LKY-7 produced no detectable amounts of lignin peroxidase, manganese peroxidase, cellulose, or xylanase under the prescribed culture conditions. A three-step chromatographic separation of the laccase indicated that at least two kinds of laccase isozymes were produced by the fungus. Activity staining of the non-boiled enzyme samples after SDS-PAGE revealed different active protein band patterns for the laccase produced in the absence or presence of the wood powder. A difference in the isozyme pattern was also observed on the activity stained gel after native PAGE. These results suggest that wood powder not only increases the production of laccase but also changes the kinds of isozymes produced by T. rubrum LKY-7. No laccase active bands were detected when the enzyme preparations were boiled for 60 s or longer, indicating that the laccase isozymes are heat-labile.

Effects of Salinity Changes on the Growth of Dunaliella salina and Its Isozyme Activities of Glycerol-3-phosphate Dehydrogenase

Dunaliella salina could survive in media containing a wide range of NaCl concentrations ranging from about 0.05 M to saturation (around 5.5 M). Glycerol is an important osmolyte when Dunaliella survive in various salt environments, and G3pdh is a key enzyme in glycerol metabolism. The osmotic response of D. salina was investigated by studying its cell growth, glycerol content change, and isozyme activity of glycerol-3-phosphate dehydrogenase (G3pdh) in different salinities. Results showed that 2.0 M NaCl was the optimal salinity for the growth of D. salina, in which condition the highest glycerol content of 64.02 +/- 3.21 (mean +/- SD) microg/mL was detected. D. salina could rapidly increase or decrease glycerol contents to adapt to hypoosmotic or hyperosmotic environments. The glycerol content declined 52.05% when salinity was changed from 2.0 to 0.5 M NaCl, and the glycerol content increased 43.61% when salinity was increased from 2.0 to 5.0 M NaCl. In the isozyme electrophoresis assay two kinds of isozymes, G3pdh and superoxide dismutase (Sod), were detected synchronously. Interestingly, it was first found that there are five isozymes of G3pdh in D. salina. G3pdh-2 mainly takes effect in moderate to high salinities, whereas the other four isozymes take effect in low salinities, which may provide an important clue for future research on osmoregulation mechanisms.

Differential Expression of EC‐SOD, Mn‐SOD and CuZn‐SOD in Rat Lung Exposed to Crystalline Silica

Superoxide dismutases (SODs) are antioxidant enzymes that catalyze the dismutation of superoxide into hydrogen peroxide. There are 3 kinds of isozymes: extracellular superoxide dismutase (EC-SOD), manganese-containing superoxide dismutase (Mn-SOD) and copper- and zinc-containing superoxide dismutase (CuZn-SOD). To examine the expression of SOD isozymes in lungs injured by crystalline silica, we intratracheally instilled male Wistar rats with 2 mg (8 mg/kg) of crystalline silica and investigated the mRNA, protein level and distribution of SOD isozymes in the rat lungs using RT-PCR, western blot analysis and immunostaining, respectively at from 3 d to 180 d of recovery following the exposure. EC-SOD mRNA levels significantly increased from 3 d to 90 d and the EC-SOD protein level was significantly higher after 90 and 180 d recovery in the crystalline silica exposed groups than in the control groups. Mn-SOD increased in silica treated rat lungs at both mRNA and protein levels, peaking at 30 d post-exposure. CuZn-SOD mRNA levels were decreased at 3, 7 and 30 d, and CuZn-SOD protein levels were also significantly lower than the control group at 90 and 180 d recovery. There was prominent EC-SOD immunostaining mainly in the plasma and alveolar macrophages and strong Mn-SOD staining in alveolar macrophages and interstitial cells of the proximal and distal portions of the alveolar duct following crystalline silica exposure. There was less CuZn-SOD staining in epithelial cells at terminal bronchioles in the crystalline silica-exposed group. These findings suggest that these SOD isozymes may be related to lung injury induced by crystalline silica.

Evidence for the existence of isozymes of glucose isomerase from Streptomyces phaeochromogenes.

Glucose isomerase from Streptomyces phaeochromogenes was purified from a commercial preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5 m, and hydroxyapatite column chromatographies. It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite. They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties. The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide. The results suggest that glucose isomerase from S. phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.