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A cytoplasmic fructose-1,6-diphosphate-dependent lactate dehydrogenase (LDH; EC 1.1.1.27) from Streptococcus mutans OMZ175 was purified to homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purification consisted of ammonium sulfate precipitation of the cytoplasmic fraction, DEAE-Sephacel and Blue-Sepharose CL.6B chromatography, and Sephacryl S200 gel permeation. The catalytic activity of the purified enzyme required the presence of fructose-1,6-diphosphate with a broad optimum between pH 5 and 6.2. The concentration of fructose-1,6-diphosphate required for half-maximal velocity was around 0.02 mM and was affected by the pyruvate concentration. The enzyme seemed to have at least two binding sites for the activator which interact in a cooperative manner. Increasing concentrations of fructose-1,6-diphosphate up to 2 mM enhanced the relative affinity of the enzyme for pyruvate and modified the pyruvate saturation curve from sigmoidal to hyperbolic. The enzyme activity showed also a sigmoidal response to NADH, exhibiting two binding sites for the cofactor with a Hill coefficient of about 1.9. The molecular weight of the native enzyme was 150,000 as determined by gel permeation on Sephacryl S200. Monomers (38,000 daltons) and dimers (85,000 daltons) were observed by sodium dodecyl sulfate-gel electrophoresis; the latter form was dissociated after reduction with 2-mercaptoethanol, and the enzyme could be considered a tetramer. Antibodies obtained against the purified S. mutans OMZ175 LDH cross-reacted with the sodium dodecyl sulfate-dissociated forms of LDHs from different S. mutans serotypes, Streptococcus sanguis OMZ9, Lactobacillus casei ATCC 4646, and Actinomyces viscosus NY 1. A competitive enzyme-linked immunosorbent assay allowed us to detect a very close relationship between the native states of L-LDHs from S. mutans serotypes and S. sanguis. Cross-reactions were also observed with the LDHs from A. viscosus and L. casei, the latter being the least related. A very weak immunological relationship was obtained between the L-LDH from S. mutans OMZ175 and the D-LDH from Lactobacillus leichmannii, whereas no cross-reaction could be detected with mammal LDHs.
The molecular weight, the amino acid composition and the N-terminal and C-terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non-allosteric (L. plantarum, L. acidophilus) L-lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined. The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the the substratebinding site of the L-lactate dehydrogenase dehydrogenase of several animals. However, the 'essential' cysteine residue 165 is replaced by threonine, as it is in the L-lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L-lactate dehydrogenases, but also the allosteric and lactate dehydrogenases from bacterial sources may have evolved from a common gene.
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The complete amino-acid sequence of L-lactate-dehydrogenase from the mesophilic Bacillus megaterium was determined. 92% of the 318 amino acids were established by sequence analysis of the N-terminus, of four CNBr fragments and of one fragment obtained by cleavage with BNPS-skatole. The primary structure was completed by sequencing overlapping fragments obtained by further cleavage of suitable CNBr fragments and BNPS fragments with either trypsin, endoproteinase Lys-C, o-iodosobenzoic acid or hydroxylamine. The C-terminal amino acids were determined by degradation with carboxypeptidase A. The sequence homology between lactate dehydrogenases from B. megaterium and those from other Bacilli is 59-61% and 35-37% to those from higher organisms. The high sequence homology among lactate dehydrogenases from Bacilli, adapted to different temperatures, allows comparative studies of the structural basis of protein thermostability.
The lactate dehydrogenase gene, ldh, of Alcaligenes eutrophus H16 was identified on a 14-kbp EcoRI restriction fragment of a genomic library in the cosmid pHC79 by hybridization with a 50-mer synthetic oligonucleotide which was derived from the N-terminal amino acid sequence of the purified enzyme. Recombinant strains of Escherichia coli JM83, which harboured a 2.0-kbp PstI subfragment in pUC9-1, expressed LDH at a high level, if ldh was downstream from and colinear to the E. coli lac promoter. The nucleotide sequence of a region of 4245 bp revealed several open reading frames which might represent coding regions. One represented the ldh gene. The amino acid sequence deduced from ldh exhibited 29% and 36% identity to the L-malate dehydrogenase of Methanothermus fervidus and to the putative translation product of an E. coli sequence of unknown function, respectively. The ldh was separated by short intergenic regions from two other open reading frames: ORF5 was located downstream of and colinear to ldh, and its putative translational product revealed 38 to 56% amino acid identity to penicillin-binding proteins. ORF3 was located upstream of and colinear to ldh, and its putative gene translational product represented a hydrophobic protein. A sequence, which resembled the A. eutrophus alcohol dehydrogenase promoter, was detected upstream of ORF3, which most probably represents the first transcribed gene of an operon consisting of ORF3, ldh and ORF5.
Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.
Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.
Lactate dehydrogenase C4 (LDH-C4) is an antigenic protein found only in spermatozoa and the mature testis. Synthetic peptides containing the amino acid sequences of the C-subunit, designated MC5-15, MC97-110 and MC211-220, were each conjugated to the carrier proteins diphtheria toxoid or bovine serum albumin. Rabbits immunized with these peptide-carrier conjugates produced antibodies to the peptide that cross-reacted with native LDH-C4. These data support our map that identifies antigenic domains of LDH-C4. Such synthetic peptides will be useful in the design of a contraceptive vaccine.
The lactate dehydrogenase (LDH) activity of mouse brain homogenates was examined after exposure to hyperbaric oxygen (5763.8 mm Hg Po2) and compared to room air controls (158.8 mm Hg Po2). The effect of reduced glutathione on LDH activity after hyperbaric oxygen exposure was also examined. The activity of LDH after treatment with hyperbaric oxygen was significantly diminished when compared with controls. In the presence of reduced glutathione, homogenates exposed to hyperbaric oxygen demonstrated higher activity than did homogenates incubated without glutathione. It is concluded that oxygen-induced inhibition occurs through the oxidation of essential free sulfhydryl groups and that this oxidation can either be prevented by reduced glutathione or the disulfide bridges may be reduced to free sulfhydryl groups by the glutathione after oxidation.
An extensive comparative structural analysis of lactate dehydrogenase (LDH) sequences from thermophilic, mesophilic and psychrophilic bacilli revealed characteristic primary structural differences. These specific amino-acid substitutions were found in the entire LDH molecule. However, in certain regions of the LDH an accumulation of these exchanges could be detected. These regions seem to be particularly important for the temperature adaptation of the enzyme. The influence of one of such regions at the N-terminus on stability and activity of LDHs was analysed by the construction of hybrid mutants between LDH sequences from thermophilic, mesophilic and psychrophilic bacilli and also by site-directed mutagenesis experiments at five different positions. The substitutions of Thr-29 or Ser-39 to Ala residues in the LDH from the mesophilic B. megaterium increased the thermostability of the enzyme drastically (15 degrees C). An increase of 20 degrees C could be observed when both amino-acid substitutions were introduced. These amino-acid substitutions resulted in an increase of Km for pyruvate and led to a three-fold reduction of the activity (kcat/Km) at 40 degrees C compared with the wild type enzyme. The influence of these amino-acid substitutions was also investigated in the LDHs from thermophilic and psychrophilic bacilli. The high heat resistance of the LDH from the thermophilic B. stearothermophilus was not altered by the Ala to Thr and Ser substitutions at positions 29 and 39, respectively. This indicates a cooperatively stabilized conformation of this LDH. However, in this mutant of the B. stearothermophilus LDH the activity (kcat/Km) was increased two-fold.
1. The behavior of H4-lactate dehydrogenase in a solution of sodium chloride was studied with respect to its molecular weight. The molecular weight decreased as the concentration of sodium chloride increased. 2. On a controlled-pore glass column equilibrated by 0.5 M soldium chloride, H4-lactate dehydrogenase was found to have mol. wt 77,500. 3. In a low salt column (0.1 M sodium chloride or less), on the other hand, the molecular weight was found to be about 135,000. 4. Salt concentration dependent association-dissociation system of the enzyme was also observed by the sedimentation equilibrium method.
The plasma level of lactate dehydrogenase (LDH) activity rises to about ten times the normal level by 4 days after infection of mice with lactate-dehydrogenase elevating virus (LDV). The levels of seven other enzymes are also increased, but to a lesser degree. SJL/J mice demonstrate a unique, genetically controlled 20-fold increase in the plasma level of LDH enzyme after LDV infection, as well as enhanced levels of the other plasma enzymes elevated by LDV infection. Comparison of virus infection in SJL/J and Swiss mice as well as in cultures of peritoneal exudate cells made from them indicated that the time course and extent of virus replication was similar for the two strains of mice. The rate of clearance of intravenously injected rabbit or mouse LDH was found to be impaired to a similar extent in LDV-infected SJL/J and Swiss mice. The effect of LDV infection on the levels of endogenous LDH released as a result of injection of carbon tetrachloride or tumor growth was also similar in the two strains of mice. These results suggest that LDV infection may specifically induce a greater influx of LDH into the plasma of SJL/J mice from an as-yet-unknown source than in other strains of mice.
Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.
The structure-function peculiarities of human, porcine, rabbit, and rat lactate dehydrogenase (LDH) isoenzymes have been studied using antipeptide antibodies (AB) against the M4-isoform of porcine LDH. Antipeptide AB were raised against the hypervariable (40% homology) N-terminal fragment (residues 1-32), and the highly conservative fragment 180-214 containing histidine in the enzyme active center. Whereas antipeptide AB against the fragment of the active center of porcine LDH M4-isoform selectively inhibited the catalytic activities of LDH isoenzymes from various sources, antipeptide AB directed against the N-end were without effect. The ability of antipeptide AB to specifically interact with various isoforms of LDH suggests that sequences 1-32 and 180-214 are immunochemically identical only in the case of human and porcine M4 isoenzymes; the relatedness of the amino acid sequence to the common antigenic determinant required the absence in the given sequence of essential amino acid substituents. Chemical modification of porcine M4-isoform by diethylpyrocarbonate and the use of specific AB revealed that histidine-195 located in the active center of LDH is not directly involved in the binding to AB.
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