• Journal of Life Science
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• v.20 no.2
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• pp.260-268
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• 2010

The lactate dehydrogenase (EC 1.1.1.27, LDH) isozymes in tissues from Channa argus were purified and characterized by biochemical, immunochemical and kinetic methods. The activity of LDH in skeletal muscle was the highest at 380.4 units and those in heart, eye and brain tissues were 13.4, 3,5 and 5.4 units, respectively. Citrate synthase (EC 4.1.3.7, CS) activity in heart tissue was the highest at 20.7 units. LDH/CS in skeletal muscle, heart, eye and brain tissues were 172.9, 0.6, 0.32 and 0.47. Protein concentration in skeletal muscle tissue was 14.7 mg/g and specific activities of LDH in skeletal muscle, heart, eye and brain tissues were 25.88, 0.79, 0.31 and 1.38 units/mg, respectively. Therefore, skeletal muscle tissue was anaerobic and heart tissue was aerobic. The LDH isozymes in tissues were identified by polyacrylamide gel electrophoresis, immunoprecipitation and Western blot with antiserum against $A_4$, $B_4$, and eye-specific $C_4$. LDH $A_4$, $A_3B$, $A_2B_2$. $AB_3$ and $B_4$ isozymes were detected in every tissue, $C_4$, $AC_3$, $A_2C_2$ and $A_3C$ were detected in eye tissue, and $A_3C$ was found in brain tissue. LDH $A_4$, $A_3B$, $A_2B_2$, $AB_3$, $B_4$, eye-specific $C_4$ isozymes were purified by affinity chromatography and Preparative PAGE Cells. The LDH $A_4$ isozyme was purified in the fraction from elution with $NAD^+$ containing buffer of affinity chromatography. Eye-specific $C_4$ isozyme was eluted right after $A_4$, after which $B_4$ isozyme was eluted with plain buffer. As a result, one part of molecular structures in $A_4$, $B_4$ and eye-specific $C_4$ were similar, but were different from each other in $B_4$ and $C_4$. Therefore the subunit A may be conservative in evolution, and the evolution of subunit B seems to be faster than that of subunit A. The activity of LDH $A_4$, $A_2B_2$, $B_4$, and eye-specific $C_4$ isozymes remained at 39.98, 21.28, 19.67 and 16.87% as a result of the inhibition by 10 mM of pyruvate, so the degree of inhibition was very high. The $Km^{PYR}$ values were 0.17, 0.27 and 0.133 mM in $A_4$, $B_4$ and eye-specific $C_4$ isozymes, respectively. The optimum pH of LDH $A_4$, $B_4$, eye-specific $C_4$, $A_2B_2$, $A_3B$, and $AB_3$ were pH 6.5, pH 8.5, pH 5.5, pH 6.0-6.5, pH 5.0 and pH 7.5. The $A_4$ and heterotetramer isozymes stabilized a broad range of pH. Especially, LDH activities in skeletal muscle tissue were high, resulting in a high degree of muscle activity.LDH metabolism in eye tissue seems to be converted faster from pyruvate to lactate by eye-specific $C_4$ isozyme as eye-specific $C_4$ have the highest affinity for pyruvate, and right after the conversion, oxidation of lactate was induced by $A_4$ isozyme. It was found that expression of Ldh-C, affinity to substrate and reaction time of $C_4$ isozyme were different according to the ecological environmental and feeding capturing patterns.

• Tuberculosis and Respiratory Diseases
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• v.41 no.4
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• pp.339-353
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• 1994

Background : The p53 gene codes for a DNA-binding nuclear phosphoprotein that appears to inhibit the progression of cells from the G1 to the S phase of the cell cycle. Mutations of the p53 gene are common in a wide variety of human cancers, including lung cancer. In lung cancers, point mutations of the p53 gene have been found in all histological types including approximately 45% of resected NSCLC and even more frequently in SCLC specimens. Mutant forms of the p53 protein have transforming activity and interfere with the cell-cycle regulatory function of the wild-type protein. The majority of p53 gene mutations produce proteins with altered conformation and prolonged half life; these mutant proteins accumulate in the cell nucleus and can be detected by immunohistochemical staining. But protein overexpression has been reported in the absence of mutation. p53 protein overexpression or gene mutation is reported poor prognostic factor in breast cancer, but in lung cancer, its prognostic significance is controversial. Method : We investigated the p53 abnormalities by nucleotide sequencing, polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP), and immunohistochemical staining. We correlated these results with each other and survival in 75 patients with NSCLC resected with curative intent. Overexpression of the p53 protein was studied immunohistochemically in archival paraffin- embedded tumor samples using the D07(Novocastra, U.K.) antibody. Overexpression of p53 protein was defined by the nuclear staining of greater than 25% immunopositive cells in tumors. Detection of p53 gene mutation was done by PCR-SSCP and nucleotide sequencing from the exon 5-9 of p53 gene. Result: 1) Of the 75 patients, 36%(27/75) showed p53 overexpression by immunohistochemical stain. There was no survival difference between positive and negative p53 immunostaining(overall median survival of 26 months, disease free median survival of 13 months in both groups). 2) By PCR-SSCP, 27.6%(16/58) of the patients showed mobility shift. There was no significant difference in survival according to mobility shift(overall median survival of 27 in patients without mobility shift vs 20 months in patients with mobility shift, disease free median survival of 8 months vs 10 months respectively). 3) Nucleotide sequence was analysed from 29 patients, and 34.5%(10/29) had mutant p53 sequence. Patients with the presence of gene mutations showed tendency to shortened survival compared with the patients with no mutation(overall median survival of 22 vs 27 months, disease free median survival of 10 vs 20 months), but there was no statistical significance. 4) The sensitivity and specificity of immunostain based on PCR-SSCP was 67.0%, 74.0%, and that of the PCR-SSCP based on the nucleotide sequencing was 91.8%, 96.2% respectively. The concordance rate between the immunostain and PCR-SSCP was 62.5%, and the rate between the PCR-SSCP and nucleotide sequencing was 95.3%. Conclusion : In terms of detection of p53 gene mutation, PCR-SSCP was superior to immunostaining. p53 gene abnormalities either overexpression or mutation were not a significant prognostic factor in NSCLC patients resected with curative intent. However, patients with the mutated p53 gene showed the trends of early relapse.