Summary
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1.
Lactate dehydrogenase (LDH), octopine dehydrogenase (ODH), and alanopine dehydrogenase (AlDH) from muscle tissues of the cockleCardium tuberculatum have been separated. Purification (ca. 100-fold) was carried out by ammonium sulfate fractionation, ion exchange chromatography, gel chromatography, and affinity chromatography.
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2.
Ion exchange chromatography and electrophoresis separated four isoenzymes of AlDH comigrating with strombine dehydrogenase (StDH) activity (Mr ca. 44 kDa). In addition, two isoenzymes of ODH (about 44 kDa) were discovered. Thed-specific LDH eluted in two separate peaks after gel filtration (about 60 and 120 kDa).
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3.
Kinetic studies on purified LDH, ODHs and AlDHs showed that the apparent Michaelis-Menten constants (K m) for NADH are quite similar in all cases (range: 0.009 to 0.048 mM). The apparentK m values for the substrate pyruvate vary from 0.047 mM (LDH II) to 0.42 mM (AlDH I with alanine as a cosubstrate) and 1.23 mM (ODH II). The apparentK m values of the ODH isoenzymes for arginine are 1.52–1.94 mM. The values of AlDHs for alanine and glycine are about 60 and 200 mM, respectively.
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4.
The apparentK m values of the substrates being oxidized are 0.94 mM (alanopine, AlDH II), 1.14 mM (octopine, ODH I), 4.62 mM (d-lactate, LDH II), 7.43 mM (octopine, ODH II) and 12.6 mM (strombine, AlDH II). The apparentK m value for NAD+ is 0.012 mM for LDH and 10 to 20-fold higher for ODH and AlDH.
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5.
Octopine formation was competitively inhibited by NAD+ (K i=0.45 mM) and octopine (K i=0.2 to 0.35 mM). The activity of AlDH is noncompetitively inhibited by propionate,d-lactate, succinate, andl-lactate (K i=0.23 mM, 1.35 mM, 2.0 mM, and 4.8 mM, respectively).
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6.
Apparent equilibrium constants of the pyruvate reducing reaction catalysed byd-LDH, ODH, AlDH, and StDH were determined.
$$K_{{\text{eq}}} = 7.35 \times 10^4 , 3.33 \times 10^5 {\text{M}}^{{\text{ - 1}}} ,10^6 {\text{M}}^{\text{1}} $$and 2.67×106 M−1, respectively.
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7.
Using the in vivo concentrations of substrates and inhibitors, the equilibrium constants, and the obtained kinetic data, an explanation for the separate function of the different dehydrogenases in the anaerobic metabolism ofC. tuberculatum is proposed.
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Abbreviations
- AlDH :
-
alanopine dehydrogenase
- EDTA :
-
ethylenediamine tetraacetic acid
- LDH :
-
lactate dehydrogenase
- ODH :
-
octopine dehydrogenase
- StDH :
-
strombine dehydrogenase
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Meinardus-Hager, G., Gäde, G. The separate function ofd-lactate-, octopine-, and alanopine dehydrogenases in the foot muscle of the jumping cockleCardium tuberculatum during anaerobiosis. J Comp Physiol B 156, 873–881 (1986). https://doi.org/10.1007/BF00694264
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DOI: https://doi.org/10.1007/BF00694264