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Endocytosis and breakdown of 125I-labelled lactate dehydrogenase isoenzyme M4 by rat liver and spleen in vivo.

ABSTRACT: 1. Porcine lactate dehydrogenase isoenzyme M4 was labelled with 125I and injected intravenously into rats. Enzyme activity and radioactivity in plasma were cleared in an identical way with a half-life of about 30 min. This half-life was the same as that of unlabelled enzyme. 2. Uptake of label by liver and spleen was determined. Radioactivity in these tissues increased up to about 13 min after injection and subsequently declined. Radioautography indicated uptake of the enzyme by sinusoidal liver cells (probably Kupffer cells) and by spleen macrophages. After differential fractionation of liver, acid-precipitable radioactivity was largely found in the light mitochondrial and microsomal fractions, suggesting localization in lysosomes and endosomes respectively. 3. The amount of acid-soluble radioactive breakdown products in plasma started to rise between 7 and 15 min after injection. Breakdown in liver and spleen was retarded by previous injection of suramin, an inhibitor of lysosomal proteolysis. 4. The contribution of liver and spleen towards the clearance of the enzyme could be calculated from its half-life in plasma and its uptake by the organs within the first 13 min period after injection. Our results indicate that about 65% and 12% of the injected dose was taken up, and subsequently broken down, by liver and spleen respectively. 5. Unlabelled porcine lactate dehydrogenase isoenzyme H4 showed a plasma half-life of about 8 h. This isoenzyme is therefore endocytosed by liver at a much slower rate than isoenzyme M4 (if it is taken up at all).

SUBMITTER: Sinke J

PROVIDER: S-EPMC1161013 | biostudies-other | 1979 Apr

REPOSITORIES: biostudies-other

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Project description:Testis-specific lactate dehydrogenase (LDH-C₄) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C₄ in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000-5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C₄ in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A₄ (LDH-A₄), Lactate Dehydrogenase B₄ (LDH-B₄), and LDH-C₄ were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C₄ for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C₄ for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A₄ (Ki = 26.900 mmol/L), LDH-B₄ (Ki = 23.800 mmol/L), and LDH-C₄ (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A₄ and LDH-B₄ were stronger than LDH-C₄. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C₄.

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Endocytosis and breakdown of 125I-labelled lactate dehydrogenase isoenzyme M4 by rat liver and spleen in vivo.

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