Structural relatedness of mouse lactate dehydrogenase isozymes,A4 (muscle), B4 (heart), and C4 (testis)

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A₄), LDH-H(B₄), and LDH-X(C₄), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.     

Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.     

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

The cDNA cloning and molecular evolution of reptile and pigeon lactate dehydrogenase isozymes

The cDNAs encoding lactate dehydrogenase isozymes LDH-A (muscle) and LDH-B (heart) from alligator and turtle and LDH-A, LDH-B, and LDH-C (testis) from pigeon were cloned and sequenced. The evolutionary relationships among vertebrate LDH isozymes were analyzed. Contrary to the traditional belief that the turtle lineage branched off before the divergence between the lizard/alligator and bird lineages, the turtle lineage was found to be clustered with either the alligator lineage or the alligator-bird clade, while the lizard lineage was found to have branched off before the divergence between the alligator/turtle and bird lineages. The pigeon testicular LDH-C isozyme was evidently duplicated from LDH-B (heart), so it is not orthologous to the mammalian testicular LDH-C isozymes.      

Expression of the cDNA encoding for mouse sperm-specific lactate dehydrogenase C in Chinese-hamster ovary cells.

The cloned cDNA encoding for mouse sperm-specific lactate dehydrogenase C (LDH-C) was inserted immediately downstream to the MMTV 5' LTR promoter, and it was shown to synthesize mouse LDH-C polypeptide in Chinese-hamster ovary cells. The mouse LDH-C subunit and the endogenous Chinese-hamster LDH-A subunit formed in vivo a heterotetrameric LDH-A3C1 isoenzyme, and this novel isoenzyme exhibited enzymic activity utilizing lactate as substrate.     

The structure of mouse testicular lactate dehydrogenase isoenzyme C4 at 2.9 A resolution.

The structure of lactate dehydrogenase isoenzyme C4 from mouse testes was solved at 2.9 A resolution using the technique of molecular replacement. The electron density map revealed a ternary-like configuration of the flexible loop peptide although density corresponding to the coenzyme and substrate molecules was not present. Apparently the apo-lactate dehydrogenase molecule in solution is in a dynamic equilibrium between the O (loop open as found in dogfish apo-lactate dehydrogenase M4) and C (loop closed as found in a variety of ternary complexes) conformations. During crystallization of the apoenzyme one or the other conformers is selected. The apparent stability of the closed conformation for the apo-lactate dehydrogenase C4 molecule may in part explain the low catalytic turnover number of the C isoenzyme. A possible substitution of an arginine residue at position 30 may also be a contributing factor as well as allowing NADP to act as coenzyme.     

Molecular cloning of cDNA sequences transcribed from mouse liver endoplasmic reticulum poly(A)mRNA

Poly(A)mRNA was isolated from the endoplasmic reticulum (ER) of male BALB/c mice and used as the template for cDNA synthesis. Double-stranded (ds) cDNA was cloned in a bacterial plasmid and 21 clones were selected for further study. Six clones have been identified by hybrid selection of mRNA, translation of the mRNA in a template-dependent system, polyacrylamide gel electrophoresis and specific immunoprecipitation. Four code for the major urinary protein of the mouse (MUP), one for serum albumin and one for alpha 1-antitrypsin. A seventh codes for an unidentified polypeptide with Mr = 27000. Initial-rate hybridisation kinetics of immobilised cloned sequences with end-labelled mRNA showed that serum albumin mRNA is most abundant in the population of mRNA species present in the endoplasmic reticulum fraction of female BALB/c liver. MUP mRNA is also very abundant. The alpha 1-antitrypsin mRNA and the mRNA specifying the 27000-dalton protein are about 5 times less abundant than serum albumin mRNA on a molar basis.     

Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.

Tektins are a class of proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules. We report here the molecular cloning of a new member of the tektin family, tektin-t, identified from a mouse haploid germ cell-specific cDNA library. Tektin-t mRNA encodes a protein of 430 deduced amino acids possessing RSNVELCRD, the conserved sequence of tektin family proteins. Western blotting showed a single band having a molecular weight of 86 kDa in the mouse testis. Immunohistochemistry of the testis showed that tektin-t is localized in the flagella of elongating spermatids from developmental step 15 to maturity.     

Molecular characterization of genetic mutations in human lactate dehydrogenase (LDH) B (H) variant

Summary We have previously detected a single base substitution of G by A at the Arg codon CGC in exon 4 of the mutant lactate dehydrogenase (LDH) gene, an unstable LDH-B variant (case 1). Here, we use the polymerase chain reaction (PCR) to amplify genomic DNA of two cases (the original case 1 and a new patient, case 2). We were able to confirm that case 1 is homozygous for the mutation, causing a replacement of the conserved Arg by His at residue 173. The resulting LDH-B variant subunit is unstable in vivo. Whereas the mutation in exon 4 was not observed in case 2, a different single base substitution of A by C was detected at the Ser codon AGT in exon 3. This mutation causes a replacement of the conserved Ser by Arg at residue 131. Genomic analysis of the family of case 2 by mismatched PCR showed that the missense mutation was consistent with their biochemical phenotypes. The replacement results in a conformational change of the residues near the Ser, probably because the side chain of Arg is much more bulky than that of Ser. The change may affect the arrangement of the cofactor binding site and result in the loss of enzyme activity. The experimental observations are consistent with computer graphics analyses.     

Molecular cloning of mouse protein kinase C (PKC) cDNA from Swiss 3T3 fibroblasts

A set of complementary DNAs (cDNAs) that encode the complete 672-amino acid sequence for mouse protein kinase C (PKC) type alpha have been isolated from a mouse Swiss 3T3 cDNA library. Extensive rescreening of this cDNA library only resulted in the isolation of clones of the same PKC species, indicating that Swiss 3T3 fibroblasts express exclusively PKC-alpha. This enzyme is encoded by two different mRNAs that exhibit a substantial heterogeneity in their 3'-noncoding regions. This heterogeneity could have been derived from alternative splicing of the pre-mRNAs or be due to differential usage of the polyadenylation motif. The expression of PKC mRNA in fibroblasts is very low and it is not influenced by treatment with the tumor promoter 12-O-tetradecanoyl-13-phorbol-acetate.     

Molecular evolution of mammalian lactate dehydrogenase-A genes and pseudogenes: association of a mouse processed pseudogene with a B1 repetitive sequence

A mouse genomic clone containing a lactate dehydrogenase-A (LDH-A) processed pseudogene and a B1 repetitive element was isolated, and a nucleotide sequence of approximately 3 kb was determined. The pseudogene and B1 element are flanked by perfect 13-bp repeats, and the B1 sequence starts at 14 nucleotides 3' to the presumptive polyadenylation signal of the pseudogene. The nucleotide sequences of the LDH-A genes and processed pseudogenes from mouse, rat, and human were compared, and a phylogenetic tree was constructed. The rate and pattern of nucleotide substitutions in the LDH-A pseudogenes are similar to previously reported results (Li et al. 1984). The average rate of nucleotide substitutions in the LDH-A pseudogenes is 4.3 X 10(- 9)/site/year. The substitutions of C----T and G----A are most frequent, and A----G substitutions are relatively high. The rate of synonymous substitutions in the LDH-A genes is 5.3 X 10(-9), which is not significantly higher than the average rate of 4.7 X 10(-9) for 35 mammalian genes. The rate of nonsynonymous substitutions in the LDH-A genes is 0.20 X 10(-9), which is considerably lower than the average rate of 0.88 X 10(-9) for 35 mammalian genes. Thus, the mammalian LDH-A gene appears to be highly conserved in evolution.      

Isolation and characterization of a mouse protein C cDNA.

Protein C (PC) is a vitamin K-dependent serine protease, a deficiency of which results in thrombus. There is no spontaneously occurring mouse model of the disease. Attempts to create such a model in mice by using anti-sense gene technology requires isolation of a normal mouse PC cDNA. When a mouse liver (BALB/c) cDNA library was screened using a human PC cDNA as a probe, nine overlapping cDNA clones were isolated and sequenced. The cloned mouse PC cDNA comprised 1,512 nucleotides and the open reading frame of the cDNA encoded a polypeptide of 461 amino acids residues including a leader peptide composed of 41 amino acids. Mouse PC exhibited high homology to both human and bovine PCs. Mouse PC also had several structural features common in other PCs; locations of 23 Cys residues, location of putative beta-hydroxy Asp71, possible carbohydrate attachment sites involving Asp residues at amino acid positions 249, 314, and 330, and location of active sites such as His212, Asp258, and Ser361. Northern blot hybridization analysis identified a single species of mouse PC mRNA (2.0 kb in length) in mouse liver.     

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.