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Data obtained from immunochemical studies and the use of allelic variants support the hypothesis that the lactate dehydrogenase isozymes unique to the teleost nervous system are encoded in a third locus. Immunochemical and other studies demonstrate that this third locus is closely related to the lactate dehydrogenase B locus.
Several binary and ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) were studied by saturation transfer ESR spectroscopy by means of an active NAD analog, spin-labeled at N6. The mobility of the spin-label depends on the nature of small molecules bound at the remote catalytic end of the coenzyme. The spin-label was found to serve as a reporter group monitoring the conformation of the peptide loop that is folded down over the active cleft in crystals of ternary complexes. The data suggest a fluctuation of the loop between open and closed forms in solution. The structure of the inhibitor molecules has been correlated with their ability to stabilize a more closed conformation of the loop.
The effect of lactate dehydrogenase (LDH) on human plaque pH and acid anion concentrations in vivo was investigated. Rinsing with sucrose solutions supplemented with LDH (1 or 2 U/ml) gave rise to reduced pH fall, decreased cH area and an increase in the mean minimum pH when compared with rinsing with sucrose only (p less than 0.05). Mean acid anion estimations showed that plaque fluid concentrations of lactate, acetate and proprionate significantly decrease (p less than 0.05) following rinsing with sucrose containing LDH at both levels and decreases in succinate when 1 U/ml LDH was present, whereas formate concentrations were only significantly lower when 2 U/ml LDH was added to sucrose rinses. It is thought that the ability of LDH to produce an alteration in the acid end-products of plaque metabolism may provide some protection against dental caries.
The allosteric effect of fructose 1,6-bisphosphate (Fru-1,6-P2) on L-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from Thermus caldophilus GK24 was studied by means of 1H NMR analyses. The conformation of NAD+ as bound to the T. caldophilus enzyme was elucidated by analyses of the transferred nuclear Overhauser effects (TRNOE), in the presence and the absence of the allosteric effector, Fru-1,6-P2. Upon binding of Fru-1,6-P2 to the enzyme, the ribose ring of the adenosine moiety of NAD+ is converted from the C2'-endo form to the C3'-endo form. This C3'-endo form of the adenosine moiety is similar to that of NAD+ as bound to nonallosteric vertebrate enzymes. However, the anti conformation of the adenine-ribose bond of NAD+ as bound to the T. caldophilus enzyme is not affected by the binding of Fru-1,6-P2. In contrast, the syn conformation of the nicotinamide-ribose bond is converted to the anti form on the binding of Fru-1,6-P2, while the ribose ring remains in the C3'-endo form as found in the case of a nonallosteric enzyme. Such a conformational change of enzyme-bound NAD+ as found on TRNOE analysis is essentially involved in the allosteric regulation of the T. caldophilus enzyme by Fru-1,6-P2.
The role of three histidine residues (His205, His296 and His303) and Asp259, important for the catalysis of NAD+-specific D-lactate dehydrogenase, was investigated using site-directed mutagenesis. None of these residues is presumed to be involved in coenzyme binding because Km for NADH remained essentially unchanged for all the mutant enzymes. Replacement of His205 with lysine resulted in a 125-fold reduction in kcat and a slight lowering of the Km value for pyruvate. D259N mutant showed a 56-fold reduction in kcat and a fivefold lowering of Km. The enzymatic activity profile shifted towards acidic pH by approximately 2 units. The H303K mutation produced no significant change in kcat values, although Km for pyruvate increased fourfold. Substitution of His296 with lysine produced no significant change in kcat values or in Km for substrate. The results obtained suggest that His205 and Asp259 play an important role in catalysis, whereas His303 does not. This corroborates structural information available for some members of the D-specific dehydrogenases family. The catalytic His296, proposed from structural studies to be the active site acid/base catalyst, is not invariant. Its function can be accomplished by lysine and this has significant implications for the enzymatic mechanism.
The fructose 1,6-bisphosphate [Fru(1,6)P2]-dependent lactate dehydrogenase in cells of Streptococcus uberis N.C.D.O. 2039 was purified by a procedure that included chromatography on DEAE-cellulose and Blue Sepharose CL-6B in phosphate buffers. The enzyme appeared to interact with Blue Sepharose through NADH-binding sites. The homogeneous enzyme had catalytic properties that were generally similar to those of other Fru(1,6)P2-dependent lactate dehydrogenases, and it had no catalytic activity in the absence of Fru(1,6)P2. Its existence in different forms, depending on conditions, was investigated by ultracentrifugation, analytical gel filtration and activity measurements. It consisted of subunits with Mr 35,900 +/- 500 and, in the presence of adequate concentrations of Fru(1,6)P2, phosphate or NADH, it existed as a tetramer, whereas when these ligands were in lower concentrations or absent, the subunits were in a concentration-dependent association-dissociation equilibrium. Dissociation occurred slowly and inactivated the enzyme, and although added ligands reversed the dissociation, the lost activity was at best only partly restored. An exception occurred when dissociation was caused by a decrease in temperature, in which case the lost activity was fully restored at the original temperature. The tetramer also lost activity at certain ligand concentrations without dissociating. The results together indicated the presence on the enzyme of two classes of binding site for both Fru(1,6)P2 and NADH, and the likelihood that phosphate bound at the same sites as Fru(1,6)P2. Two different ligands together were much more effective at preventing inactivation and dissociation than was expected from their effectiveness when present separately. It was concluded that tetrameric forms of the enzyme rather than the enzyme in association-dissociation equilibrium were involved in the regulation of its activity in vivo.
Circulating enzymes may be inactivated in the plasma and the inactive breakdown products may be hydrolyzed in the lumen of the small intestine. Evidence for this mechanism was based upon previous studies with 125I-labeled lactate dehydrogenase-5, and here similar studies with radioiodinated lactate dehydrogenase-1 are reported, to determine whether this isoenzyme is similarly catabolized. The pure rabbit enzyme was labeled with 125I by use of lactoperoxidase and hydrogen peroxide (the labeled enzyme had 80-85% of the original catalytic activity). After its intravenous injection into rabbits, plasma enzyme activity and radioactivity disappeared during the first 4 h at similar fast rates, apparently because of distribution of the injected enzyme throughout the extracellular fluid. During a second phase (30-h), catalytic activity disappeared significantly faster than radioactivity, suggesting inactivation of the enzyme in either the plasma or a compartment in close proximity to it, or both. Enzyme activity then remained constant while plasma radioactivity continued to decrease at a slower, exponential rate, apparently owing to removal of breakdown products. In no case did tissue radioactivity, studied 6 h after injection, approach that of plasma. We therefore conclude that removal of the enzyme protein or its breakdown products is a passive process. Appreciable radioactivity was detected in the intestinal contents, a finding which suggests that removal via the small intestine is an important route for the removal of inactivated enzyme products from the circulation. Less than 3% of the injected radioactivity appeared in the feces during the first three days; urinary excretion accounted for about 67% during the same period, about 60% of which consisted of radio-iodinated amino-acids, the remainder of iodide. Free mono- and di-iodotyrosines were among the products excreted. These appear to originate from absorption of the products of further breakdown of the enzyme molecule in the intestine.
We developed a new convenient method for determination of magnesium ion concentrations by use of magnesium ion-dependent enzymes; pyruvate kinase and lactate dehydrogenase. This method is based on the determination of the reaction rate of pyruvate kinase which uses MgADP- as substrate. The reaction rate of pyruvate kinase is dependent upon the formation of the complex with ADP and magnesium ion and the amount of the complex is dependent upon that of magnesium ion. The reaction rate of pyruvate kinase can be easily and spectrophotometrically determined by using lactate dehydrogenase and NADH as the decreased amount of absorbance at 340 nm.
L-Lactate dehydrogenase (LDH) cDNAs encoding for LDH-A(4) (muscle) and LDH-B(4) (heart) isozymes from caiman (Caiman crocodilus apaporiensis) belonging to the order Crocodilia and Chinese soft-shelled turtle (Pelodiscus sinensis) belonging to the order Chelonia were sequenced. The phylogenetic relationships of the newly determined cDNA and their deduced protein sequences, as well as the previously published sequences of vertebrate LDH isozymes, were analyzed by various phylogenetic tree construction methods. These results indicated that Chelonia is indeed more closely related to Crocodilia. The divergent times between caiman and alligator, turtle and soft-shelled turtle, and Chelonia and Crocodilia were estimated to be approximately 36, 100 and 177 million years, respectively.
The Diagnostic Validity of Enhanced Lactate Dehydrogenase Activity in the Vaginal Secretion for Early Diagnosis of Diseases of the Female Genitals and the Mamma in Need of Treatment. An impregnated hygiene tampon (Gynaegnost) was used, which by a colour reaction permits the determination of augmented activity of lactate dehydrogenase (LDH) in vaginal secretion. The diagnostic validity of the results of the findings by this method in the detection of diseases of the female genitals and the mamma needing treatment was examined in two clinical diagnostic studies. The first study including 486 patients chiefly suffering from manifest carcinoma was aimed at solving a deliberately limited question. In patients with carcinoma stages I to IV of the most important genital locations augmented LDH-activity (correct positive results) was found in about 90%. A second study comprised 1955 women entering into outpatient gynaecological services for preventive examination; the definition of this series was difficult. Three categories of diagnostic groups can be determined by the results of this study. In the first category with the most important subgroup "routine examination without specification of a pathological finding" less than 20% "false" positive test results by the indicator were observed. A second category with inflammatory diseases (colpitis, endometritis) showed positive results in about one-third of the examinations. The third category comprised a minor number of malignant tumors roughly with a percentage of positive results corresponding to that of the first study. Augmented LDH activity in vaginal secretion is a diagnostic criterion which detects not only cancer; it is similar to the test of occult blood in faeces.(ABSTRACT TRUNCATED AT 250 WORDS)
Histochemical and electrophoretic examinations of the effect of Biseptol 480 on lactate dehydrogenase in the rat heart muscle were carried out on white male rats of Wistar strain of about 300 g in weight. The rats were divided into 2 groups: experimental group of 10 rats and a control one of 5 rats. Experimental rats were given, by stomach tube, 80 g of Biseptol 480, suspended in 1 cm3 of distilled water for 7 consecutive d. Control rats were given 1 cm3 of distilled water for 7 consecutive d too. Samples were taken from the experimental and control rats to examine lactate dehydrogenase and to make isoenzyme separation. The lack of differences between the experimental rat samples and those control ones permitted to conclude that Biseptol 480 administered to rats, did not bring about significant histoenzymatic and electrophoretic changes suggesting a disbalance in the examined stage of metabolism in the rat heart muscle.
In an attempt to identify glycolytic capacity of normal and neoplastic human ovary, total lactate dehydrogenase (LDH) activity was measured in tissue cytosol originating from 69 patients (18 with benign ovarian tumor, 34 with ovarian carcinoma, six with nonepithelial ovarian malignant tumors, and 11 with tumor metastatic to ovary) and compared to the LDH activity of normal ovarian tissues (n = 19). Median value of total LDH-specific activity expressed as U/mg protein was 0.546 in normal tissues, 0.584 in benign tumors, 1.071 in malignancies metastatic to ovaries, 0.872 in nonepithelial primary ovarian tumors, and 0.818 in primary carcinomas. A significant rise in LDH-specific activity was found in malignant primary and secondary tumors of epithelial and nonepithelial origin, but not in benign neoplasms, compared to the activity in normal tissue. Ovarian carcinomas of serous histologic type did not differ in LDH activity from mucinous tumors. However, poorly differentiated carcinomas (grade 3) showed significantly enhanced activity of this glycolytic enzyme when compared to its grade 1 counterpart. The subgroup of grade 1 tumors did not differ in LDH activity from normal and benign ovarian tissue. Obtained results suggest that direct correlation might exist between ovarian epithelial tumor grade and lactate dehydrogenase activity.
Treatment of cancers with cytotoxic agents such as alkylating drugs often, but not always results in transient to permanent testicular dysfunction. The present study was planned to investigate the effects of dacarbazine [5-(3,3-dimethyltriazeno) imidazole-4-carboxamide] on testicular function in mice. Swiss albino mice (9-12 weeks old) were treated with 0, 5, 25, 50, or 100mg/kg body weight/day dacarbazine (i.p.) for 5 days at intervals of 24h between treatments. Mice were sacrificed on days 7, 14, 21, 28, 35, 49, and 70 after the last treatment (6 mice/dose/sample time), and the epididymal sperm count, sperm motility, sperm morphology, testicular histopathology (qualitative histopathology, seminiferous tubular diameter and epithelial height), and intra-testicular levels of testosterone and lactate dehydrogenase were assessed. Dacarbazine decreased the body weight only on day 28 at 25mg/kg dose-level, but increased the paired testes weights at 50mg/kg on day 7, at 25-100mg/kg on day 14, and at 25 and 50mg/kg on day 21 (P<0.05-0.01; one-way ANOVA and Bonferroni's post hoc test). The sperm count was decreased on all sampling days except at 5 and 25mg/kg dose-levels on day 70, but with severe oligospermia on days 28 and 35 (P<0.05-0.001). The sperm motility was decreased at 100mg/kg on days 14 and 21, at 5, 25, and 100mg/kg on day 28, and at all dose-levels on day 35 (P<0.05-0.001). Dacarbazine induced both head and tail abnormalities and some sperms with cytoplasmic droplets, but significant increase was seen in all dose groups on days 14 and 21, and at 100mg/kg dose-level on day 35. Drug-induced epithelial sloughing was seen on days 14-35 and other histopathological changes observed were vacuoles and abnormal cells. The STD was increased at 25-100mg/kg on day 7, at all dose-levels on day 14, at 50-100mg/kg on days 21 and 28, but without any effects on days 35-70 (P<0.05-0.001), and the tubular lumen was found dilated. The SE was increased on days 7, 21 and 28 at 100mg/kg and on day 14 at 50-100mg/kg. Dacarbazine reduced the intra-testicular testosterone level at 100mg/kg on day 7, at 5, 50 and 100mg/kg on day 14, at all dose-levels on days 21, 28, and 35, and at 50mg/kg on day 49 (P<0.05-0.001). The intra-testicular lactate dehydrogenase concentration increased at all dose-levels up to day 35, but without any effect on days 49 and 70 (P<0.05-0.001). There was no particular dose-response of dacarbazine on any parameters tested. The sperm count (except on day 7-positive correlation; Pearson product moment correlation) or sperm motility did not have any relation but increase in abnormal sperms showed negative correlation with decrease in testosterone level on days 7, 21 and 28. Decrease in sperm count was in negative correlation on days 14 and 35, and increase in abnormal sperms showed positive correlation on day 35 with increase in LDH level. Finally, the decrease in sperm motility had no correlation with increase in abnormal sperm shapes. We conclude that dacarbazine is genotoxic and cytotoxic to the mouse testis in a transient fashion, and these effects are exerted along with decrease in testosterone and increase in lactate dehydrogenase levels in the testis.
While within a human host the opportunistic pathogen Toxoplasma gondii relies heavily on glycolysis for its energy needs. Lactate dehydrogenase (LDH), the terminal enzyme in anaerobic glycolysis necessary for NAD(+) regeneration, therefore represents an attractive therapeutic target. The tachyzoite stage lactate dehydrogenase (LDH1) from the parasite T. gondii has been crystallized in apo form and in ternary complexes containing NAD(+) or the NAD(+)-analogue 3-acetylpyridine adenine dinucleotide (APAD(+)) and sulfate or the inhibitor oxalate. Comparison of the apo and ternary models shows an active-site loop that becomes ordered upon substrate binding. This active-site loop is five residues longer than in most LDHs and necessarily adopts a different conformation. While loop isomerization is fully rate-limiting in prototypical LDHs, kinetic data suggest that LDH1's rate is limited by chemical steps. The importance of charge neutralization in ligand binding is supported by the complexes that have been crystallized as well as fluorescence quenching experiments performed with ligands at low and high pH. A methionine that replaces a serine residue and displaces an ordered water molecule often seen in LDH structures provides a structural explanation for reduced substrate inhibition. Superimposition of LDH1 with human muscle- and heart-specific LDH isoforms reveals differences in residues that line the active site that increase LDH1's hydrophobicity. These differences will aid in designing inhibitors specific for LDH1 that may be useful in treating toxoplasmic encephalitis and other complications that arise in immune-compromised individuals.
Three families with a complete deficiency of the lactate dehydrogenase M subunit show exertional myoglobinuria. The response to ischemic forearm work is characteristic in these three families: an increase of venous lactate concentration after ischemic work was not observed and a marked increase of venous pyruvate was found. Glycolysis was markedly retarded in the patient's muscle in the glyceraldehyde 3-phosphate dehydrogenase (GA3PD) step. A significant increases in glyceraldehyde 3-phosphate, dihydroxyacetone phosphate and fructose 1,6-diphosphate were observed. The glycolysis retardation may be attributed to the impaired reoxidation of NADH produced by GA3PD action. The cytosolic fraction of skeletal muscle is rich in alpha-glycerophosphate dehydrogenase. This enzyme reoxidizes the excess NADH and drains triose phosphates from the glycolytic pathway under anaerobic conditions. For this reason, ATP production was significantly impaired and muscle cells were damaged in these patients. Consequently, the cytosolic enzymes and proteins such as creatine kinase and myoglobin were released into the blood stream. Otherwise, patients with a lactate dehydrogenase M-subunit deficiency do not show muscle stiffness and myoglobinuria under ordinary circumstances. They complain of muscle rigidity and sudden myoglobinuria after strenous exercise under anaerobic conditions. Thus, the lactate dehydrogenase M-subunit deficiency does not show any symptoms under ordinary circumstances, but is a latent hereditary disorder, now recognized as a new type of hereditary exertional myoglobinuria.
The paper deals with the effects of the gravitational field investigated either by hypokinesia with corresponding diminution of muscle solicitation, or by centrifugation, that is by rise of gravitational solicitation, on lactate dehydrogenase activity of rats. After 7 days of hypokinesia the activity of lactate dehydrogenase is significantly changed by the elevation of LDH5 isoenzyme, both in serum and in myocardium. The enhancement is by 40% in serum and by 30% in myocardium against the control group. In centrifuged rats the enhancement of LDH5 isozyme is much lesser than in hypokinetic rats, that is by 18% in myocardium and by 20% in serum against the control group. The total of all LDH1-5 isozymes was found in cardiac muscle of both hypokinetic, centrifuged and control group. In serum only two fractions LDH2 and LDH5 isozymes appear both in hypokinetic, centrifuged and control group.
Testicular lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH) activity were measured at 1 and 4 hr following intratesticular injection of morphine and dynorphin. Twenty five and 50 micrograms doses of morphine sulfate significantly reduced LDH activity at 1 hr after injection. Five and 25 micrograms doses of dynorphin reduced LDH activity both at 1 and 4 hr after treatment. Testicular SDH activity was increased by morphine at 1 hr followed by a decrease at 4 hr. Both doses of dynorphin significantly reduced SDH activity at 1 and 4 hr after treatment. These results indicate paracrine regulatory role for opioids in testicular metabolism.
In cultured epithelial cells of rat liver the isoenzyme patterns of pyruvate kinase, lactate dehydrogenase and alkaline phosphatase were studied and compared with those of freshly isolated parenchymal and non-parenchymal liver cells. In all epithelial cell lines pyruvate kinase was not activated by fructose 1,6-bisphosphate, suggesting the absence of the L-isoenzyme. Cell lines derived from livers of newborn rats expressed LDH-4 and -5, whereas cell lines developed from fetal rat livers contained all 5 lactate dehydrogenase isoenzymes. In the latter case the pattern was found to depend on the state of confluence. All cell lines exhibited only a single alkaline phosphatase form, however, differences were found with respect to electrophoretic mobility.