Effects of phosphate on the dissociation and enzymic stability of rabbit muscle lactate dehydrogenase

Lovell, Stephen J.; Winzor, Donald J.

doi:10.1021/bi00714a018

Cited by 34 publications

(17 citation statements)

References 26 publications

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“…Protection of LDH activity by glutathione is consistent with the presence of cysteine residues in the LDH molecule. LDH from rabbit muscle is a tetrameric protein with a molecular weight of about 140,000 (Lovell and Winzor, 1974). A monomer of LDH contains six cysteine residues out of a total of 571 amino acids (www.expasy.org, primary accession number Q8X666).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies

Kendig

Tarloff

2007

Toxicology in Vitro

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Lactate dehydrogenase (LDH) release is frequently used as an end-point for cytotoxicity studies. We have been unable to measure LDH release during studies using para-aminophenol (PAP) in LLC-PK 1 cells. When LLC-PK 1 cells were incubated with either PAP (0-10 mM) or menadione (0-1000 μM), viability was markedly reduced when assessed by alamar Blue or total LDH activity but not by release of LDH into the incubation medium. In addition, we incubated cells with PAP or menadione and compared LDH activity using two different assays. Both assays confirmed our observation of decreased LDH activity in cell lysates without corresponding increases in LDH activity in incubation media. Using purified LDH and 10 mM PAP, we that PAP produced loss of LDH activity that was inversely proportional to the amount of LDH initially added. In additional experiments, we incubated 0.5 units of LDH for 1 h with varying concentrations of PAP, menadione, hydrogen peroxide (H 2 O 2 ) or cisplatin. All four chemicals produced concentration-dependent decreases in LDH activity. In previous experiments, inclusion of antioxidants such as reduced glutathione (GSH) and ascorbate protected cells from PAP toxicity. GSH (1 mM) preserved LDH activity in the presence of toxicants while ascorbate (1 mM) only prevented LDH loss induced by PAP. These studies suggest that LDH that is released into the incubation medium is susceptible to degradation when reactive chemicals are present.

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Section: Discussionmentioning

confidence: 99%

Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies

Kendig

Tarloff

2007

Toxicology in Vitro

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“…At 1000 bar the stability limit of the enzyme in the acidic pH range is found to be similar to the one observed at normal pressure. This is caused by the denaturation of the native quaternary structure at pH < 5.0 [24].…”

Section: Discussionmentioning

confidence: 99%

Dissociation and Aggregation of Lactic Dehydrogenase by High Hydrostatic Pressure

Schmid

Lüdemann

Jaenicke

1979

European Journal of Biochemistry

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As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and -SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Liidemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90-98; Euv. J. Biochem. 86, 21 9 -2241. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation ( 5 10 min) at pH 6.0-8.5 and pressures of 0.3 -1 .O kbdr causes dissociation which is reversed at normal pressure. At 5 "C the activation volume is found to be AV* = -62 2 3 cm3 . mol-'. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5 "C is A V* = -97The results may be described by a reaction sequence comprising pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive highmolecular-weight particles. 3 cm3 . mol-'.

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“…Therefore, we decided to check whether the interfacing areas can play the key role in CELLULAR & MOLECULAR BIOLOGY LETTERS 391 complex formation with anionic liposomes in the case of skeletal muscle LDH. The M 4 isoform of LDH from rabbit skeletal muscles was chosen for the experiments because it has been evidenced that this enzyme is stabilized in its tetrameric form at acidic pH (around 5.0) by NADH/NAD coenzyme [33]. The performed experiments proved that there is no difference between the interaction of the two muscle enzymes with acidic phospholipids in the presence and in the absence of the cofactor.…”

Section: Resultsmentioning

confidence: 99%

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Terlecki

Czapińska

Hotowy

2007

Cellular and Molecular Biology Letters

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Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids.

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Effects of phosphate on the dissociation and enzymic stability of rabbit muscle lactate dehydrogenase

Cited by 34 publications

References 26 publications

Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies

Inactivation of lactate dehydrogenase by several chemicals: Implications for in vitro toxicology studies

Dissociation and Aggregation of Lactic Dehydrogenase by High Hydrostatic Pressure

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

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