Lactate Dehydrogenase Turnover Research Articles

How to draw kinetic barrier diagrams for enzyme-catalysed reactions

A modified way to construct kinetic barrier diagrams is presented. Although the diagram superficially resembles a free-energy profile, it is independent of any conception derived from transition-state theory. Some simple calculations referring to the lactate dehydrogenase turnover reaction at equilibrium demonstrate self-consistency of the diagram and its direct relevance to the results of numerical simulations of the detailed course of enzyme-catalysed reactions.

A study of lactate dehydrogenase levels and turnover rates during postnatal development in the rat

Specific radioimmunoassays for lactate dehydrogenase A and B subunits have been employed to quantify cellular contents of these proteins more precisely than hitherto possible and to monitor changes during postnatal development. Liver, skeletal muscle, heart muscle and kidney cortex all demonstrated alterations in cellular levels of lactate dehydrogenase subunits over the first 56 days of life, the particular pattern being specific to each tissue. Studies on the turnover of lactate dehydrogenase in vivo and in vitro indicated that the developmental changes in total lactate dehydrogenase content in liver and kidney were regulated at some point(s) during both the biosynthesis and the degradation of the proteins.

The turnover of lactate dehydrogenase in skeletal muscle of the mouse: Influence of fibre type and exercise regimen

1. 1. The influence of fibre-type and an exercise regimen on the turnover characteristics of proteins in mouse skeletal muscle has been studied. 2. 2. Exercise induced a weight increase in most individual muscle and a decrease in lactate dehydrogenase activity which was more marked in white muscle than red. The coincident decrease in M-subunit activity was more noticeable in fast-twitch muscles than in slow-twitch types. 3. 3. Exercise caused a considerable and consistent increase in the rate of degradation of total soluble proteins in individual muscles, with turnover being higher in muscle with a high red fibre content. 4. 4. The response of lactate dehydrogenase to exercise varied considerably between individual muscle as indicated by the comparative rate constants for synthesis and degradation. In general, degradation was greather in muscle with a high slow-twitch or red fibre content. 5. 5. Overall, a considerable redistribution of protein resources was observed during this physiological perturbation (exercise) with the main emphasis away from the pool of total soluble protein towards the myofibrillar constituents. Highly individualistic responses in relation to rates of synthesis and degradation were observed for the specific protein, lactate dehydrogenase, in the separate muscle of this animal.

The influence of ehrlich ascites tumour growth on the turnover of lactate dehydrogenase in tissues of the mouse

1. 1. The influence of Ehrlich ascites tumour growth on the turnover of total soluble protein and lactate dehydrogenase (LDH) in mouse tissues has been studied. 2. 2. Turnover parameters were determined by means of double-labelling technique, with the enzyme (LDH) being isolated by affinity chromatography. 3. 3. Tumour growth was accompanied by a decreased rate of synthesis of total protein in all tissues. 4. 4. Lactate dehydrogenase by contrast snowed an increased rate of synthesis in all tissues but kidney. 5. 5. These directions of change, in combination with the lesser response of degradation constants, resulted in a consequent conservation of enzyme activity in all tissues except kidney. 6. 6. A generalized shift in the LDH isozyme pattern of these tissues was also observed during tumour growth with an increased contribution of A-type subunit. 7. 7. These results have been discussed in relation to the redirection of protein synthesis and degradation, the occurrence of foetal isozymes, and possible mechanisms involved in the redistribution of protein resources in the animal during tumour development.

Effect of pregnancy on the turnover of lactate dehydrogenase and its isoenzymes in mouse tissues

The turnover characteristics of total protein and of lactate dehydrogenase and its isoenzymes have been determined in several tissues of the mouse during the later stages of pregnancy. Whereas most tissues showed markedly decreased rates of both synthesis and degradation of proteins, the incorporation rate in uterus increased. Significant differences in the extent of isotope distribution between total protein and lactate dehydrogenase and its isozymes were also noted. Overall the concept emerges of a pool of body protein which is responsive to the physiological stresses of pregnancy at the level of both tissue and cellular function.

The effect of thiamine deficiency on the turnover of lactate dehydrogenase in mouse tissues

1. 1. Groups of mice were subjected to different degrees of thiamine deprivation in their diet. In particular, the effects of complete thiamine deficiency and a continuation of minimal nutritional levels of thiamine were compared. 2. 2. The effects of these treatments on the turnover characteristics of lactate dehydrogenase and total soluble protein have been studied by means of double labelling experiments, and determinations of the relative emphases of synthesis and degradation of these tissue components. 3. 3. Marked divergences from normal were apparent with each of these nutritional regimens-complete thiamine deficiency causing a considerably increased rate of degradation for both total protein and lactate dehydrogenase in all tissues; whereas maintenance of minimal levels of thiamine led to increased degradation of total protein in liver, but reduced rates of degradation for lactate dehydrogenase in brain, heart and liver. 4. 4. The significance of these results has been discussed in relation to the relative influence of vitamin and calorie deficiencies on turnover parameters, the individuality of specific tissue behaviour, differences in protein redistribution in response to separate physiological perturbations, and the role of thiamine in specific proteolysis.

The effect of starvation and protein depletion on the turnover of lactate dehydrogenase in mouse tissues

1. 1. The influence of protein depletion and starvation on the turnover parameters of total soluble protein and lactate dehydrogenase in mouse tissues has been studied. 2. 2. Animals on a 20% protein diet reached a new steady state after 2 weeks, and this was characterized by a generalized decrease in the rates of both degradation and synthesis. 3. 3. Some tissues (e.g. kidney) were less affected than others (e.g. muscle, brain), and lactate dehydrogenase was more highly conserved than total protein, in general. 4. 4. Markedly different turnover characteristics were observed in starvation, with increased degradation of total protein being common in the terminal stages. 5. 5. These results have been discussed in relation to the individuality of the tissue responses and possible intracellular mechanisms.

Regulation of lactate dehydrogenase levels in the mouse.

BERNARDO NADAL-GINARD From the Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461 The number of lactate dehydrogenase molecules per cell equivalent in normal adult mouse tissues is: heart muscle 1.13 x 10s, skeletal muscle 3.6 x 1O8, liver 3.94 x lo’, and kidney 3.91 x 107. These steady state levels of lactate dehydrogenase reflect a balance between synthesis and degradation. To study the relative role of the two processes the half-lives of lactate dehydrogenase in these four mouse tissues were determined by means of a combination of the double isotope labeling technique and enzyme purification by oxamate . aminohexyl . Sepharose 4B affinity chromatog- raphy. This technique makes possible the measurement of lactate dehydrogenase turnover in single mouse organs and allows for the comparison of isotope decay between different tissues without being affected by differences in the size of the amino acid pools. All the lactate dehydrogenase isozymes in the same tissue were found to have the same half-life. The half-lives were: heart 8.2 days, skeletal muscle 43.0 days, liver 4.3 days, and kidney 6.1 days. The rates of lactate dehydrogenase synthe- sis in these tissues were calculated to be 110, 67, 74, and 52 molecules per cell per s, respectively. From these data and from the proportion of A and B subunits in each tissue, the rates of synthesis of each subunit were calculated. From the results it is concluded that the different steady state lactate dehydrogenase levels, a phenotypic expression of the lactate dehydrogenase genes, reflect mainly the differ- ing rates of degadation since the rates of synthesis are very similar; thus the regulation of the quantitative expres- sion of the lactate dehydrogenase genes in the mouse is largely post-translational.

Ontogenic characteristics of lactate dehydrogenase turnover in the mouse

1. 1. In order to investigate the ontogenic and turnover characteristics of lactate dehydrogenase ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27) in the foetal mouse, activities of this enzyme and isoenzyme distributions have been measured during development, and the time sequence of incorporation of radioactive amino acids have been determined during the last weeks of gestation. 2. 2. In the early foetal stages, the enzyme was present with low specific activity and in the form of lactate dehydrogenase-5 in all regions, but a marked increase in specific activity became evident about the seventeenth day of gestation, and was accompanied by the formation of tissue specific patterns of isoenzyme distribution. 3. 3. Measurement of turnover parameters was undertaken by both double-label ([ 3H] and [ 14C]leucine) and pulse-chase ([ 3H]leucine) techniques. Both procedures provided indications that appreciable degradation may occur during embryogenesis, with general proteolysis proceeding at a faster rate than with lactate dehydrogenase, and with higher rates of degradation occurring in foetuses which were smaller than average. 4. 4. This data has been discussed in relation to the regional variation in these characteristics and the significance to studies of growth and differentiation.

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates.

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

Macroscopic rate constants involved in the formation and interconversion of the two central enzyme--substrate complexes of the lactate dehydrogenase turnover.

The preceding paper (Südi, 1974) reports partial success in describing the conversion of E(NADH) plus pyruvate into E(NAD+) plus lactate in terms of a simple Haldane-type scheme which involves two intermediates (E(NADH) (Pyr) and E(NAD+) (Lac)), where E represents lactate dehydrogenase. This information is completed here by reporting kinetic results obtained by carrying out the same reaction in the opposite direction. The combined results of these two papers confirm the findings of Holbrook & Gutfreund (1973) that the observed spectral changes do take place at the level of resolution of this simple two-intermediate scheme. The following numerical values for the rate (and equilibrium) constants involved in their formation and decomposition are reported: [Formula: see text] It is shown that although the precision of estimation of some of these numerical values is subject to some experimental uncertainty, their derivation from direct experimental observations only involves the principle of microscopic reversibility. This paper describes stopped-flow kinetic observations made with E(NAD+) and lactate as the two reactants. It is shown that fluorescence and u.v.-absorption measurements yield the same experimental rate constant for the last reaction step in which E(NADH) is generated. On the other hand, the generation of E(NADH) (Pyr) can only be indirectly observed, as a less than stoicheiometric ;burst', and by u.v.-absorption measurements only. It is shown that the stoicheiometry of this partial ;burst reaction', and a pre-equilibrium factor in the directly observed rate of E(NADH)-production, yield equivalent information about the reversible oxidation-reduction step. It is further shown that the pre-equilibrium factor that is involved in the generation of E(NADH) can be determined because k(+4)=222s(-1) is already known (Südi, 1974). Since the fluorescence measurements yield much more precise estimations, and their interpretation is considered by the author to be free of ambiguity, the presented quantitative analysis is based on the fluorescence observations.

Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers.

Histochemical, biochemical, and contractile properties of red, white, and intermediate fibers.

Studies on Rates of Abortive Ternary Complex Formation of Lactate Dehydrogenase Isozymes

Abstract Rates of formation and dissociation of an abortive ternary complex between lactate dehydrogenase isozymes from various mammalian tissues, NAD+, and pyruvate were studied utilizing stopped-flow spectrofluorometry. Formation of the complex was accompanied by a decrease in protein fluorescence at 340 mµ and a loss of lactate dehydrogenase activity. Rates of formation of an abortive lactate dehydrogenase-1 and lactate dehydrogenase-5 complex were compared at 25° and 40° over a range of pyruvate and enzyme concentrations, at two NAD+ concentrations, and with two coenzyme analogues. Dialysis against buffer produced an increase in protein fluorescence and recovery of lactate dehydrogenase activity that suggested dissociation of the abortive complex. At pyruvate concentrations of 0.3 mm the abortive ternary complex formed twice as fast with lactate dehydrogenase-1 as with lactate dehydrogenase-5 and with 40.0 mm pyruvate it formed 5 times faster. The rate of complex formation increased with both isozymes when pyruvate concentrations were raised from 0.3 mm to 40.0 mm and when the temperature was elevated from 25° to 40°. However, the rates of abortive complex formation were inversely related to enzyme concentration. Compared to lactate dehydrogenase turnover numbers, the rates of formation of the abortive ternary complex were slow. Under physiological conditions of temperature (40°) and pyruvate concentration (0.3 to 1.0 mm), half the maximum fluorescence quenching required approximately 50 sec for partially purified lactate dehydrogenase-1. Changes in coenzyme concentration from 0.10 mm to 0.75 mm failed to alter the rates of abortive complex formation. With the 3-acetylpyridine analogue of NAD+, rates of abortive complex formation for both lactate dehydrogenase-1 and lactate dehydrogenase-5 were similar, whereas with deamino-NAD+ the abortive complex formed faster with lactate dehydrogenase-1 than with lactate dehydrogenase-5. Dialysis of the abortive complex resulted in recovery of enzyme activity; the rate of recovery was faster with lactate dehydrogenase-5 than with lactate dehydrogenase-1. The stopped-flow spectrophotofluorometer permitted measurement of lactate dehydrogenase activity at enzyme concentrations approaching those in tissue. At lactate dehydrogenase-1 concentrations of 3.5 x 10-6 m or 3.5 x 10-7 m no substrate inhibition was detected with pyruvate concentrations up to 20.0 mm, whereas with enzyme levels at and below 1.8 x 10-7 m, substrate inhibition appeared at pyruvate concentrations greater than 1.0 mm.