Purification and partial biochemical characterization of recombinant lactate dehydrogenase 1 (LDH-1) of the white shrimp Litopenaeus vannamei.

Lactate dehydrogenase (LDH) is a key enzyme to produce energy during hypoxia by anaerobic glycolysis. In the white shrimp Litopenaeus vannamei, two protein subunits (LDH-1 and LDH-2) were previously identified, deduced from two different transcripts that come from the same LDH gene by processing via mutually exclusive alternative splicing. LDH-1 contains exon five and LDH-2 contains exon six and the two proteins differ only in 15 amino acid residues. Both subunits were independently cloned and overexpressed in E. coli as a fusion protein containing a chitin binding domain. Previously, recombinant LDH-2 was successfully purified and characterized, but LDH-1 was insoluble and aggregated forming inclusion bodies. We report the production of soluble LDH-1 by testing different pHs in the buffers used to lyse the bacterial cells before the purification step and the characterization of the purified protein to show that the cDNA indeed codes for a functional and active protein. The recombinant native protein is a homotetramer of approximately 140 kDa composed by 36 kDa subunits and has higher affinity for pyruvate than for lactate. LDH-1 has an optimum pH of 7.5 and is stable between pH 8.0 and 9.0; pH data analysis showed two pKa values of 6.1 ±â€¯0.15 and 8.8 ±â€¯0.15 suggesting a histidine and asparagine, respectively, involved in the active site. The enzyme optimal temperature was 44 °C and it was stable between 20 and 60 °C. LDH-1 was slightly activated by NaCl, KCl and MgCl2 and fully inhibited by ZnCl2.

White shrimp Litopenaeus vannamei recombinant lactate dehydrogenase: Biochemical and kinetic characterization.

Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pKa of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants Km for NADH was 23.4 ± 1.8 µM, and for pyruvate was 203 ± 25 µM, while Vmax was 7.45 µmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia.

Valor pronóstico de las isoenzimas de LDH en pacientes con linforma no hodgkin / Prognostic value of LDH isoenzymes in patients with non-Hodgkin linforma

La enzima lactato deshidrogenasa (LDH) es un factor pronóstico en Linfoma No Hodgkin (LNH). El objetivo del trabajo consistió en evaluar prospectivamente el valor pronóstico de las isoenzimas de LDH en pacientes con LNH. Se estudiaron 67 pacientes de primera consulta con diagnóstico de LNH, sin tratamiento previo, VIH negativo y sin otras enfermedades, tiempo promedio de seguimiento 30 meses (rango 3-48 meses). Las muestras de suero se recolectaron previas al tratamiento. La LDH total (LDHT) e isoenzimas de LDH se determinaron respectivamente por método cinético y electroforesis de proteínas en gel de agarosa. Se procesaron muestras de 122 controles sanos para establecer los valores de referencia de las isoenzimas de LDH. 49(73%) LNH agresivos y 18(27%) LNH indolentes y según el Índice Pronóstico Internacional (IPI), 60 (90%) bajo riesgo y 7(10%) alto riesgo. Las isoenzimas LDH1, LDH2, LDH3, LDH4 y LDH5 presentaron niveles absolutos significativamente elevados en 25 (37%), 29 (43%), 32 (48%), 20 (39%) y 11 (16%) de los casos respectivamente (p<0,0001). La actividad porcentual de LDH4 en los pacientes con LNH agresivos fue significativamente superior respecto al grupo de LNH indolentes (p=0,01). En el análisis univariado, valores absolutos elevados de LDH1 se asociaron significativamente con una sobrevida global disminuida (p=0,0064) en el grupo total de pacientes. LDH1 conservó su valor pronóstico aún en el grupo de pacientes con valores normales de LDHT (p=0,04). En pacientes con LNH agresivos, valores elevados de LDHT e IPI alto riesgo se asociaron significativamente con una menor sobrevida global (p<0,05). En el análisis multivariado la LDHT e IPI resultaron factores pronósticos independientes de la sobrevida. Alteraciones específicas del patrón de isoenzimas de LDH sugieren la relación de LDH4 con la biología del tumor y su actividad proliferativa en LNH agresivos y el valor pronóstico de LDH1 como factor adverso de la sobrevida en el análisis univariado.

Lactate dehydrogenase (LDH) is a prognostic factor in non-Hodgkin lymphoma (NHL). Our objective was to evaluate prospectively the prognostic value of LDH isoenzymes in patients with NHL. We studied 67 newly diagnosed NHL patients, previously untreated, HIV-negative and free from other disease, median follow-up of 30 month (range 3-48 month). Before starting treatment serum samples were collected for the determination of total LDH (LDHT) and LDH isoenzymes that were respectively assayed by kinetic method and protein electrophoresis in agarose gel. In order to set reference values of LDH isoenzymes samples from122 healthy controls were processed. Results: 49(73%) of the patients were aggressive NHL and 18(27%) indolent NHL and according to the International Prognostic Index (IPI), 60(90 %) low risk and 7(10%) high risk. High absolute values of LDH1, LDH2, LDH3, LDH4 and LDH5 isoenzymes were significantly elevated in 25 (37%), 29 (43%), 32 (48%), 20 (39%) and 11 (16%) of cases respectively (p<0,0001). The percentage value of LDH4 activity in aggressive NHL patients was significantly higher compared to indolent NHL group (p=0,01). In univariate analysis increased LDH1 absolute values were significantly associated with decreased overall survival in the total group of patients (p = 0.0064). LDH1 remained a prognostic factor for survival even when considering the group of patients with normal serum LDHT values (p = 0.04). In patients with aggressive NHL increased values of LDHT and high risk IPI were significantly associated with decreased overall survival (p<0.05). In a multivariate analysis LDHT and IPI score were independent prognostic factor for survival.

Purificaçö parcial da desidrogenase lática eritrocitária / Partial purification of erythrocyte lactate dehydrogenase

A purificaçäo parcial da desidrogenase lática (LDH) de glóbulos vermelhos humanos vencidos foi efetuada utilizando-se, basicamente, a DEAE-celulose equilbrada em tampäo fosfato 10mM, pH 7,6, em que a hemoglobina näo se liga, mas a desidrogenase lática se adere à resina. A eluiçäo foi feita em gradiente fosfato 10-100mM e as fraçöes onde as atividades de LDH estavam presentes foram reunidas, dialisadas e novamente submetidas a cromatografia e eluiçäo. A preparaçäo final mostrou ausência de piruvato quinase e das enzimas da glicólise, com exceçäo de negligível contaminaçäo com monofosfogliceromutase e glutationa peroxidase, também presentes na preparaçäo Sigma L2881 obtida de coraçäo de suíno

Adaptative features of enzymes from family Sciaenidae--III. Studies on lactate dehydrogenase (LDH) of fishes from the south coast of Uruguay.

1. Electrophoretic analyses of lactate dehydrogenase isozyme patterns of three species of temperate fish belonging to family Sciaenidae (order Perciformes) indicates that at least three LDH loci--Ldh-A, Ldh-B and Ldh-C are active. 2. The subunits encoded by these loci occurred at different levels in the different tissues and organs analyzed. 3. The products of these loci show different response to changes in temperature. The results were obtained by electrophoretic analyses. 4. Thermostability of skeletal muscle, heart and eye lactate dehydrogenase incubated at 60 degrees C for different periods of time were examined. 5. Relative activities of LDH isozymes were compared by Klebe's method to determine pattern of divergence of duplicated gene expression in the three species studied.

Electric organ lactate dehydrogenase:physical and kinetic properties of the purified enzyme from Electrophorus electricus (L.).

L(+)lactate dehydrogenase (LDH) from the electric organ of Electrophorus electricus (L.) was purified by ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. Purified LDH was homogeneous when examined by polyacrylamide gel electrophoresis under nondenaturing conditions. Both LDH activity and protein were demonstrable in the same band. The mobility of the LDH-5 isozyme is characteristic of the muscle type enzyme. Isoelectric focusing showed a single molecular species of pIO 6.5 +/- 0.4. The apparent molecular weight was 140,000 (+/- 10%) on the basis of gel filtration of Sephadex G-200. The effect of organic acids on the enzyme activity towards pyruvate (NADH) and lactate (NAD) was determined spectrophotometrically at 340 nm. Sodium oxamate behaved as a mixed inhibitor when lactate (NAD) was the substrate, whereas ethyl oxamate was an uncompetitive inhibitor. Both the sodium salt and the ester of oxamic acid were competitive inhibitors when pyruvate (NADH) was the substrate.