Antipeptide antibodies to the porcine lactate dehydrogenase isoenzyme M4 active center fragment as a probe for the structural analysis of active centers of human lactate dehydrogenase isoforms

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.     

Antigenic activity of porcine muscle lactate dehydrogenase fragment 180-214 containing the histidine residue of the isoenzyme active center

Solid phase immunoenzymatic analysis was used to study the antigenic activity of proteolytic degradation products of the porcine muscle lactate dehydrogenase isoform M4. The presence in the enzyme structure of topographic (linear) antigenic determinants was demonstrated. Peptide 180-214 containing histidine-195 in the active center of lactate dehydrogenase was isolated from the tryptic hydrolysate of the carboxymethylated enzyme. This peptide interacts with antibodies against the native enzyme, i.e., antibodies bound to the immunoadsorbent, and causes a 20-25% inhibition of the antigen-antibody complex formation. Protein modification by fluorescein mercuriacetate at Cys-165 essential for the enzyme activity does not result in the synthesis of antibodies that would stimulate the inhibition of the lactate dehydrogenase catalytic activity as compared to antibodies to the native isoenzyme. The putative role of some amino acid residues in the structure of antigenic determinants of porcine muscle lactate dehydrogenase is discussed.     

Comparative binding of lactate dehydrogenase to mitochondrial fractions

Beef liver mitochondrial fraction showed LDH activity (1.76 +/- 0.25 U/g pellet). Sixty seven% of the initial mitochondrial pellet LDH activity (almost M4 isoenzyme) was released when suspended in NaCl 0.15 M. When the washed particles were sonicated in a 0.15 M NaCl medium, the solubilized LDH activity (all five isoenzymes as cytosoluble fraction) was 5-fold higher than the initial pellet activity. The different isoenzymatic composition of intramitochondrial and externally bound forms of the enzyme should be taken into account when investigating the physiological role of intramitochondrial LDH. Beef liver cytosoluble LDH (very little content of M4 isoenzyme) showed no affinity for the beef liver mitochondrial fraction but purified M4-LDH isoenzyme was able to bind to the particulate fraction from the same source. This suggests an isoenzyme specificity for the interaction. The maximum amount of cytosoluble LDH bound to the mitochondrial fraction depends on the enzyme and the particulate fraction source. Therefore, binding capacity to the mitochondrial fraction depends not only on the net charge of LDH isoenzymes, which play a predominant role in the binding, but also on individual characteristics of the LDH isoenzymes and mitochondrial fractions from different sources. This suggests that electrostatic forces are not the only ones involved in the binding process.     

Energy enzymes of the flight muscle of the house sparrow, Passer domesticus--III. Comparative physical properties of heart and flight muscle lactate dehydrogenase

Purification of heart (LDH-4) and flight muscle (LDH-2 and LDH-3) lactate dehydrogenase isoenzymes from the house sparrow, Passer domesticus, has been accomplished. Although these isoenzymes electrophoretically migrate reversed to most other vertebrate LDH isoenzymes, comparison of the amino acid compositions of LDH-4 and LDH-2-LDH-3 fails to reveal the basis for their reversed electrophoretic migration. Amino acid compositions did reveal mol. wts between 141,000-142,000 as well as vp of 0.744 ml/g (LDH-4) and 0.745 ml/g (LDH-2-LDH-3). SDS-gel electrophoresis yielded single bands for each preparation with mol. wts of 35,000 suggesting that LDH in this species exists as a tetramer. LDH-4 has a lower Km for both pyruvate (0.005 mM) and NADH (0.002 mM) than does LDH-2-LDH-3 (0.062 mM for pyruvate, 0.013 mM for NADH).     

The tentative amino acid sequencing of lactate dehydrogenase C4 by X-ray diffraction analysis.

A tentative amino acid sequence of mouse testicular lactate dehydrogenase C4 was deduced from an electron density map and comparison with five other known lactate dehydrogenase sequences. The amino acid composition determined by chemical analysis agrees reasonably well with the present results. Necessary changes in amino acids were largely conservative and confined to the external portions of the molecule. Residues in the Q and P subunit contact regions were particularly well conserved as were most internal residues. The minimum base change/codon was similar between the C and H isoenzymes and between the C and M isoenzymes.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

On intratetrametric catalytic independency of active sites of lactate dehydrogenase isoenzymes]

Interrelations of active sites of tetrameric molecules of human lactatedehydrogenase (LDH), known as intratetrameric catalytic independency of subunits, are studied. Estimation of catalytic activity of subunits, which compose hybrid LDH isoenzymes, is carried out. Ratios of molecular activity of subunits are calculated and a conclution is drawn on the catalytic independency of LDH isoenzymes active sites with respect to substrate inhibition by L-lactate. Possible mechanisms of substrate inhibition of LDH isoenzymes and their inactivation with urea in the view of different interrelations of active sites of these isoenzymes under conditions studied are discussed.     

Sub-banding and fine structure of serum lactate dehydrogenase isoenzymes induced by sulfur compounds

Human serum was incubated 1:1 by volume with solutions of several sulfur compounds of pH 7.2 for at least 24 hours and the total LDH-P determined and the distribution of LDH isoenzymes and fine structure examined by polyacrylamide disc electrophoresis. As with penicillamine, the disulfide but not N-acetyl-penicillamine, caused removal of LDH-5 without affecting the total unitage. The latter was depressed by thiophenoxyacetate (0.10-0.40 M), undiluted dimethyl sulfoxide and levamisole (0.025 M and higher) but not by the last agent at 0.0005-0.001 M which gave rise to fine structure in the gels. As screened in a number of sera of 160–2300 U/L in total LDH, phthalyltetrathioacetate (0.2-1.0 M) elicited 3 unique sub-bands as pre-LDH-2, pre-LDH-3 and pre-LDH-4 to the exclusion of any effect on the total LDH.     

The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies

Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.     

Estimation of correlation of lactate dehydrogenase subunits mole quota based on differences in substrate inhibition

Summary A kinetic method of estimating the mole quota ratios of the human lactate dehydrogenase (LDH) H and M subunits based on differences in substrate inhibition of LDH isoenzymes by lactate is proposed. Stability of kinetic constants for a prolonged period of time is demonstrated. The dependence of the activity ratios on the contribution of the mole quota of the M-subunit of LDH is studied under conditions of low and high substrate concentrations. The experimental and theoretical values show the following correlation: r = 0.998; p < 0.001. A comparison of the method proposed with the electrophoretic method of LDH subunit estimation is made, the values obtained being in good agreement. No effect of the components of human diploid cell homogenate and only an insignificant effect of the blood serum components on the kinetic constants of LDH isoenzymes are shown. The applicability of the method to the estimation of the quantitative content of both LDH subunits in natural samples is demonstrated. The informational value of the method is compared to that of other standard methods of LDH isoenzyme estimation.The need of the rapid and reliable method for determination the lactate dehydrogenase (LDH) activity of the H and M subunits has long been a matter of great importance, since the study of LDH isoenzymes is an indispensable part of clinical, genetical, cytological and herontological investigations.In 1960 PLAGEMANN et al. ¹ ,making use of different substrate inhibition of H₄ and M₄ isoenzymes LDH, developed a method for the estimation of the percent composition H and M subunits LDH within any given mixture of them. The method involves the assay of mixture of LDH isoenzymes in the presence of two different levels of pyruvate. The authors calculated the percent of each subunit in a mixture from the ratio of enzymatic activities at both high and a low concentration of pyruvate. Although this method was subsequently improved, both experimentally^2–S and theoretically⁶, its application was still impossible without first eliminating a great many problems. The problem of subunit interactions inside the enzyme molecule has not been settled. In addition, questions have not been raised about stability of the kinetic parameters', the reproducibility of the method, its applicability to the study of different objects and also the informational value of the experimental data.In our previous investigation^7,8, we have studied the kinetic properties of five purified isoenzymes of human lactate dehydrogenase and demonstrated the catalytic independence of the active sites of the LDH tetrameric molecules with respect to substrate inhibition.In the present report an attempt has been made to develop a kinetic method for the assay of M-polypeptide chains mole quotum of lactate dehydrogenase in natural specimens.     

Lactate dehydrogenase: inhibition of subunit A by the sulfhydryl reagent AgNO3.

Lactate dehydrogenase isoenzymes including the subunit A--LDH 5 (A4), LDH 4 (A3B), LDH 3 (A2B2), and LDH 2 (AB3)--are inhibitable by the sulfhydryl reagent AgNO3, while the isoenzyme LDH 1 (B4) is not.     

Characterization of rabbit lactate dehydrogenase-M and lactate dehydrogenase-H cDNAs. Control of lactate dehydrogenase expression in rabbit muscle

Two cDNA clones were isolated, one corresponding to the mRNA coding for lactate dehydrogenase-M (LDH-M), the other to the mRNA coding for lactate dehydrogenase-H (LDH-H). The cDNA inserts consist of the entire open reading frame for LDH-M and a partial sequence, from amino acid 117 to 332, for LDH-H. Using these two clones as probes we demonstrate that: (a) the abundance of mRNA is muscle-type dependent; (b) the ratio M/H subunit for protein and mRNA is well related in the muscles studied; and (c) the M + H mRNA level is not relative to the total LDH activity.     

Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.     

The excretion of lactate dehydrogenase in human urine after ingestion of aspirin

1. Cells present in normal human urine contain 5-10% of the total lactate dehydrogenase excreted. The enzyme released from these cells by ultrasonication contained a distribution of isoenzymes similar to that found in the bulk of the urine and it is suggested that these cells are the main source of urinary lactate dehydrogenase. 2. Cells were thoroughly washed before examination so it is unlikely that the enzyme found in urinary sediment was simply adsorbed. In addition, full recoveries of added lactate dehydrogenase isoenzymes LDH(1) and LDH(5) showed that adsorption did not occur. 3. Most of the cells in normal urine are of the non-squamous epithelial type and their excretion is greatly increased after the ingestion by the subject of 3g. of aspirin. The possible origin of these non-squamous cells from the kidney is discussed. 4. Starch-block electrophoresis and relative activity measurements of lactate dehydrogenase excreted after the subject had taken aspirin show that the enzymes present in urine and cells are very similar, confirming the conclusion reached above (point 1). They have slightly more M subunits than the normal, shown particularly as an increase in isoenzyme LDH(2). The isoenzyme pattern is like that of the kidney medulla and the possible reasons for this are discussed in terms of the concentration of salicylic acid in various parts of the kidney. 5. The results confirm the previous suggestion that the kidney is the main source of urinary lactate dehydrogenase.     

Changes in lactate and malate dehydrogenase isoenzymes in salmonella under the effect of potassium dichloroisocyanurate

The method of enzyme-electrophoresis in agar gel according to Wieme (1959) was used for the study of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes of 24-hour and 48-hour Salmonella cultures exposed to a 0.02% solution of potassium dichloroisocyanurate (PDIC). Severe repression of LDH and MDH isoenzymes was observed immediately after the exposure of the culture to the disinfectant solution. A significant decrease in the content of the isoenzyme LDH1 and of the cytoplasmic fraction (C1) of MDH simultaneously with the appearance of the fractions LDH4, LDH1a and LDH1b were established in the strains cultured on MPA in the course of 24 hours following the exposure. A tendency to a decrease in the LDH1 content was preserved in the experimental cultures after 48 hours, but the spectrum of MDH isoenzymes showed almost no differences in comparison with that of MDH isoenzymes in 48-hour cultures of the control strains.     

The Alcaligenes eutrophus ldh structural gene encodes a novel type of lactate dehydrogenase

Abstract The lactate dehydrogenase gene, ldh , of Alcaligenes eutrophus H16 was identified on a 14-kbp Eco RI 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 Pst I 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.