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The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.
We found in a prospective study of lactate dehydrogenase isoenzyme 6 (LD6) in human tissues obtained at autopsy that LD6 was usually present in liver when death was preceded by prolonged hypotension or impaired ventilation but not in cases of sudden death. Other organs containing LD6 were kidney and spleen. LD6 is heat stable, differs from H-subunit-containing LD isoenzymes by pyruvate resistance, differs from M-subunit-containing LD isoenzymes by immunoprecipitation, and is distinct from spermatic LDX. LD6 from liver extracts acted without lactate as substrate and could be enhanced by ethanol added to the substrate. These results indicate that LD6 is not a true lactate dehydrogenase, and that it frequently appears in severe liver injury.
Salmonid fish, as a result of total genome duplication, have two genes, Ldh H and Ldh H', coding for polypeptides H and H', respectively, both of which have been shown in their tetrameric forms to be immunologically related to the classical H-4 lactate dehydrogenase isozyme of higher vertebrates (Bailey, G. S., and Wilson, A. C. (1968) J. Biol. Chem. 243,5843). The H-4 and H'-4 isozymes have now been highly purified from quinnat salmon, and their chemical, physical, immunological, and catalytic properties examined, and compared to the M-4 isozyme of salmon. The two proteins H-4 and H'-4 are shown to be very similar in amino acid composition, but significant differences in a few residues suggest differences in amino acid sequences. This suggestion was born out by quantitative immunological experiments in which the H-4 and H'-4 isozymes were shown to be about as different from each other as are the H-4 lactate dehydrogenases of chicken and duck. This suggests that the gene duplication event in salmon which give rise to two Ldh H genes occurred approximately 80 to 100 million years ago. The H'-4 lactate dehydrogenase which has risen from this duplication in salmon is shown to be somewhat intermediate between H-4 and M-4 in thermal stability, and in all catalytic properties examined, including substrate optima, Michaelis constants, and susceptibility to inhibition by high levels of substrate. In particular the H'-4 isozyme is almost exactly intermediate between H-4 and M-4 in its resistance to product inhibition by lactate, the catalytic parameter suggested to be of major functional importance to M-4 lactate dehydrogenase isozymes (Stambaugh, R., and Post D. (1966) J. Biol. Chem. 241,1462). Further, tissue distribution of these isozymes in salmon and trout are shown to be unusual. The M-4 isozyme salmon and trout are shown to be unusual. The M-4 isozyme occurs in very few tissues in detectable levels. It is the H-4 and H'-4 rather than H-4 and M-4, which occur in independently variable but significant levels in most tissues examined. Thus the H'-4 isozyme, despite its very close structural similarity to H-4 appears to possess functional properties which are different from either H-4 or M-4 in salmon, and some properties are midway between the two. This finding, together with the unusual tissue distribution of these isozymes, suggests that salmon with H'-4 lactate dehydrogenase is evolving to function catalytically in the absence of a balanced H-4-M-4 isozyme complement in most tissues. This balance seems to be met in most tissues by combinations of H-4 and H'-4,
We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)-B4 with antiacetylated LDH-B4 Fab'-horseradish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B4 concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B4 concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.
A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield.
A series of mutant strains of Lactococcus lactis were constructed with lactate dehydrogenase (LDH) activities ranging from below 1% to 133% of the wild-type activity level. The mutants with 59% to 133% of lactate dehydrogenase activity had growth rates similar to the wild-type and showed a homolactic pattern of fermentation. Only after lactate dehydrogenase activity was reduced ninefold compared to the wild-type was the growth rate significantly affected, and the ldh mutants started to produce mixed-acid products (formate, acetate, and ethanol in addition to lactate). Flux control coefficients were determined and it was found that lactate dehydrogenase exerted virtually no control on the glycolytic flux at the wild-type enzyme level and also not on the flux catalyzed by the enzyme itself, i.e. on the lactate production. As expected, the flux towards the mixed-acid products was strongly enhanced in the strain deleted for lactate dehydrogenase. What is more surprising is that the enzyme had a strong negative control ( CLDHJF1 =-1.3) on the flux to formate at the wild-type level of lactate dehydrogenase. Furthermore, we showed that L. lactis has limited excess of capacity of lactate dehydrogenase, only 70% more than needed to catalyze the lactate flux in the wild-type cells.
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Lactate dehydrogenase levels in tears were measured in normal subjects and in patients with retinoblastoma. When specimens were collected without trauma in normal subjects, there were usually no detectable levels of lactate dehydrogenase. When the eyelids were rubbed (probably liberating epithelial cells) lactate dehydrogenase levels were detectable and were five to ten times those of normal aqueous humor. When lactate dehydrogenase isoenzymes in such specimens were analyzed, the level of lactate dehydrogenase 5 was always higher than that of lactate dehydrogenase 1 (similar to normal aqueous humor). While occasional patients with retinoblastoma do have elevated levels of lactate dehydrogenase in tears, it is uncertain if this is due to epithelial destruction or retinoblastoma itself. Lactate dehydrogenase in tears does not appear to be a useful test for the diagnosis of retinoblastoma given present techniques for collection and analysis.
We assessed the clinical efficacy of a thin-layer agarose electrophoresis assay for lactate dehydrogenase isoenzyme estimation in the diagnosis of acute myocardial infarction. From a population of 228 patients admitted to the Coronary Care Unit with suspected acute myocardial infarction, all 101 positive cases (confirmed by clinical presentation and electrocardiographic changes) were correctly identified with lactate dehydrogenase isoenzyme-1 percentage of total activity above the reference range and with a lactate dehydrogenase isoenzyme-1/isoenzyme-2 ratio of 0.76 or above. This ratio was between 0.45 and 0.74 for 250 healthy subjects. No falsely negative, but 12 falsely positive results were obtained from this Coronary Care Unit population, to give a sensitivity of 100% and a specificity of 90.5% for the ratio test.
Antipeptide antibodies produced towards N-terminal site of lactate dehydrogenase-5 (LDH5) from pig muscular tissue exhibited specific reactions with N-terminal site of pig LDH5 and the whole enzyme molecule. An immunosorbent containing the antipeptide antibodies produced bound and precipitated all the LDH isoforms involving even a single M-subunit of the enzyme (LDH2, LDH5) but did not precipitate H4-isoform (LDH1) in extracts of human heart and skeletal muscle tissues and in blood serum of patients with myocardium infarction. The data obtained suggest that antipeptide antibodies against N-terminal site of pig muscular tissue LDH reacted selectively only with M-subunits of human LDH and did not interact with H4 isoform, which occurred due to dissimilarities in primary structure of N-terminal sites of M4 and H4 LDH isoenzymes.
Antipeptide antibodies (AB) to the fragment of the active center of porcine lactate dehydrogenase M4 isoform were used for the analysis of antigenic properties and structural comparison of active centers of human lactate dehydrogenase isoforms. Selective precipitation of the M-subunit-containing isoforms using an immunoadsorbent based on antipeptide AB as well as selective inhibition of the enzymic activity of the M4 isoform by antipeptide AB testify to the specific binding of isoforms to antipeptide AB. The experimental results confirm the literary data on conformational changes in the structure of the active centers of corresponding human lactate dehydrogenase isoforms. The specific interaction of antipeptide AB with human lactate dehydrogenase isoforms suggests that the site of the amino acid sequence (residues 180-214) in both human and porcine M4 isoenzymes is immunochemically identical. The data obtained suggest that antipeptide AB are convenient probes for detecting differences (including minor ones) in the primary and spatial structure of enzymes.
An electrophoretic variant of lactate dehydrogenase (LD) M(A) subunit was discovered in a female patient with chest pain. Her LD activity in serum was within the normal reference interval, and analysis of her LD isoenzyme pattern showed an abnormal migration indicating a fast-type LD-M(A) subunit variant. DNA analysis of the mutant LD-M gene detected a single base substitution, an A to G transition at codon 220 (AAA-->GAA). This mutation resulted in the replacement of a lysine by a glutamic acid (mutation K220E) and produced a subunit variant (electrophoretic fast type). This missense mutation was also observed in the patient's son, and genotypes of mother and son were consistent with their biochemical phenotypes, as evaluated by LD isoenzyme analysis.
In 385 patients with acute myocardial infarction, lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes were determined electrophoretically 24, 48, and 72 h after admission. At those times, LD-1/LD-2 ratios exceeding 1 were recorded in 78.9, 88.8, and 92.2% of the cases, respectively. LD-1 ranged from 181 to 2674 U/L, or 21.9 to 66.1% of the total activity. On the first day of hospitalization, 27.3% of the patients demonstrated abnormal LD-5 (greater than 6% of total LD); this finding dropped to 20.5% and 17.4% in the two following days. Early increases in LD-5 were most frequently observed in patients associating inferior infarcts with posterior or lateral extension and having a previous history of myocardial infarction. On day 1, LD-5 was significantly increased in early deceased patients as compared to long-term survivors (9.7% vs 4.9% of total LD, p less than 0.01). LD-5 definitely contributes to the prognostic efficiency of total LD in acute myocardial infarction, but does not replace it as a risk predictor. This study confirms the superiority of total LD over the isoenzyme measurements to achieve short-term prognostication.
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Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.
Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.
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