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We demonstrated that 5-vinyldeoxyuridine ((V)U) and 5-carboxyvinyldeoxyuridine ((CV)U) can be used to photoligate a longer oligonucleotide (ODN) from smaller ODNs on a template. By performing irradiation at 366 nm, these artificial nucleotides make photoligated ODNs with high efficiency without any side reactions. Moreover, by performing irradiation at 312 nm, these photoligated ODNs were reversed to the original ODN. (V)U needs to be irradiated 366 nm for 6 h, but (CV)U needs to be irradiated at 366 nm for 15 min. Finally, we made a self-assembled structure with an ODN containing (CV)U and observed the photoligated ODN by photoirradiation.

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC). This study evaluated the meaning of increased PDK4 mRNA in glucose metabolism by investigating PDK4 protein levels, PDC activity and glucose uptake by the heart and skeletal muscle of JVS mice. PDK4 protein levels in the heart and skeletal muscle of fed JVS mice were increased in accordance with mRNA levels, and protein was enriched in the mitochondria. PDK4 protein was co-fractionated with PDC in sucrose density gradient centrifugation, like PDK2 protein; however, the activities of the pyruvate dehydrogenase complex (PDC) active form in the heart and skeletal muscle of fed JVS mice were similar to those in fed control mice. Fed JVS mice showed significantly higher glucose uptake in the heart and similar uptake in the skeletal muscle compared with fed control mice. Thus, in carnitine deficiency under fed conditions, glucose was preferentially utilized in the heart as an energy source despite increased PDK4 protein levels in the mitochondria. The preferred glucose utilization may be involved in developing cardiac hypertrophy from carnitine deficiency in fatty acid oxidation abnormality., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: It is not known if the dihydrolipoamide succinyltransferase (DLST) gene, a mitochondrial protein, undergoes alternative splicing. We identified an uncharacterized protein reacting with an anti-DLST antibody in the I bands of myofibrils in rat skeletal muscle., Methods: Immunocytochemical staining with an anti-DLST antibody, the purification and amino acid sequence analysis of the protein, and the isolation and sequencing of the protein's cDNA were carried out to clarify the properties of the protein and its relationship to the DLST gene., Results: A pyrophosphate concentration >10 mM was necessary to extract the protein from myofibrils in the presence of salt with a higher concentration than 0.6 M, at an alkaline pH of 7.5-8.0. The protein corresponded to the amino acid sequence of the C-terminal side of DLST. The cDNAs for this protein were splicing variants of the DLST gene, with deletions of both exons 2 and 3, or only exon 2 or 3. These variants possessed an open reading frame from an initiation codon in exon 8 of the DLST gene to a termination codon in exon 15, generating a protein with a molecular weight of 30 kDa., Conclusions: The DLST gene undergoes alternative splicing, generating the protein isolated from the I bands of myofibrils., General Significance: The DLST gene produces two different proteins with quite different functions via alternative splicing.

Dihydrolipoamide succinyltransferase (DLST) is a subunit enzyme of the alpha-ketoglutarate dehydrogenase complex of the Krebs cycle. While studying how the DLST genotype contributes to the pathogenesis of Alzheimer's disease (AD), we found a novel mRNA that is transcribed starting from intron 7 in the DLST gene. The novel mRNA level in the brain of AD patients was significantly lower than that of controls. The truncated gene product (designated MIRTD) localized to the intermembrane space of mitochondria. To investigate the function of MIRTD, we established human neuroblastoma SH-SY5Y cells expressing a maxizyme, a kind of ribozyme, that specifically digests the MIRTD mRNA. The expression of the maxizyme specifically eliminated the MIRTD protein and the resultant MIRTD-deficient cells exhibited a marked decrease in the amounts of subunits of complexes I and IV of the mitochondrial respiratory chain, resulting in a decline of activity. A pulse-label experiment revealed that the loss of the subunits is a post-translational event. Thus, the DLST gene is bifunctional and MIRTD transcribed from the gene contributes to the biogenesis of the mitochondrial respiratory complexes.

The dihydrolipoamide succinyltransferase (DLST) gene of the alpha-ketoglutarate dehydrogenase complex (alpha-KGDC) was isolated from a rat genomic DNA library and sequenced. This gene was composed of 15 exons and 14 introns like the human DLST gene. Sequence analysis of the promoter-regulatory region of the rat DLST gene-(Dlst) showed the possible presence of a CAAT box-sequence and of the sequences for an AP-2 site and three Sp1 sites, but no TATA box-sequence was evidenced. The nucleotide sequences of introns 1 and 4 of the rat Dlst were significantly homologous to those of introns 1 and 4 of the human DLST gene. The sequence analysis of the rat Dlst suggested that the exon coding for the E3- and/or E1-binding domain may have been lost from the gene during evolution in eukaryotic DLST, possibly after mitochondrial symbiosis because prokaryotic DLST possesses the E3- and/or E1-binding domain.

Dihydrolipoamide succinyltransferase (E2, EC 2.3.1.61, chromosome 14q24.2-3) is a specific subunit of human alpha-ketoglutarate dehydrogenase complex (KGDHC). A biallelic intragenic polymorphism was identified in E2 gene of KGDHC. It was a single nucleotide substitution between G (in allele 1) and A (in allele 2) at the position that does not change amino acid code. Using this intragenic polymorphism as a marker, we investigated the association between this gene and Parkinson's disease. Frequencies of the genotypes that carry allele 2 were significantly higher in the Parkinson's disease group than in the control group. The results indicated that a genetic variant of the E2 gene itself or in close proximity to the gene constitutes one of the genetic risk factors for Parkinson's disease.

Dihydrolipoamide succinyltransferase (DLST) is the core-enzyme of 2-oxoglutarate dehydrogenase complex which is located in mitochondria. In this study, several tissues from rat and human were immunostained with an affinity-purified anti-DLST antibody. Of the tissues examined, the plasma membrane of skeletal muscle was immunostained with the antibody besides mitochondria. Furthermore, subcellular fractionation analysis coupled with Western blotting demonstrated that the antigen of the anti-DLST antibody is distributed on the plasma membrane fraction in addition to the mitochondria fraction in skeletal muscle and that it is free from the complex. The molecular weight of the polypeptide bound to the plasma membrane was about 20 kilodaltons (kDa). The polypeptide was purified by immunoprecipitation and its N-terminal amino-acid sequence was determined. The amino-acid sequence exactly corresponded to a part of DLST. Northern blots revealed the presence of mRNA corresponding to the 20 kDa protein. We are the first to report that a mitochondrial protein is also present on the plasma membrane in skeletal muscle as well as in mitochondria., (Copyright 1997 Academic Press.)

The distribution of three mitochondrial enzymes: dihydrolipoamide succinyltransferase, dihydrolipoamide acetyltransferase, and beta-subunit of ATP synthase, were examined in the nucleus basalis of Meynert, substantia nigra, locus coeruleus, hippocampus, and cerebral cortex of monkey brain by immunocytochemical staining. Dihydrolipoamide succinyltransferase and dihydrolipoamide acetyltransferase had parallel distribution in the substantia nigra, but dihydrolipoamide acetyltransferase was rich in the locus coeruleus, nucleus basalis of Meynert, and especially in the hippocampus in comparison with dihydrolipoamide succinyltransferase. The ATP synthase beta-subunit was strikingly rich in many neurons of the locus coeruleus and cerebral cortex in comparison with dihydrolipoamide acetyltransferase and dihydrolipoamide succinyltransferase. These results show that these mitochondrial enzymes are not expressed synchronously in the neurons of brain, suggesting the differential regulation of mitochondrial enzymes and the heterogeneity of mitochondria.

In the present study, the dihydrolipoamide succinyltransferase gene of the 2-oxoglutarate dehydrogenase complex was isolated from a human genomic DNA library and its entire nucleotide sequence was determined. This gene was approximately 23 kbp in size with 15 exons and 14 introns. All of the donor and acceptor splice sites of this gene conformed to the GT/AG rule. A guanine residue 43 bases upstreams of the ATG initiating translation codon was the transcription initiation site of the human dihydrolipoamide succinyltransferase mRNA. Sequence analysis of the promoter-regulatory region showed the presence of a CAAT-box-like sequence but the presence of a TATA-box-like sequence was not evidenced. Also located in this region were sequences resembling glucocorticoid-responsive and cAMP-responsive elements, and an Sp1 binding site. No nucleotide sequence corresponding to the E3-binding and/or E1-binding domain was found in any region of the gene. Therefore, the exon coding for the E3-binding and/or E1-binding domain may have been lost from the gene during evolution. Moreover, a processed pseudogene of dihydrolipoamide succinyltransferase was isolated and sequenced. The nucleotide sequence of the pseudogene is 93% similar to the sequence of the human dihydrolipoamide succinyltransferase cDNA, but the pseudogene is not functional for base changes, deletions and insertions of the pseudogene. Southern-blot analysis showed the presence of a single copy of this gene and a single copy of a pseudogene in the human genome. In addition, a possible relationship between dihydrolipoamide succinyltransferase and familial Alzheimer's disease is discussed.

We report an immunohistochemical study of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC) in the substantia nigra in Parkinson's disease. The KGDHC, the three enzyme complex catalyzing the oxidation of alpha-ketoglutarate to succinate through succinic semialdehyde, is the rate-regulating enzyme of the TCA cycle. The mitochondrial toxin, MPP+, inhibits not only complex I but also the KGDHC. Therefore, we investigated this enzyme complex in Parkinson's disease. In the control patients (n = 6), the immunostaining of the melanized nigral neurons was generally uniform; most of the melanized neurons showed good immunostaining with some neurons showing somewhat reduced staining. In Parkinson's disease (n = 9), many melanized neurons showed reduced immunostaining for the KGDHC, and those neurons were more frequently seen in the lateral one-third of substantia nigra. The decrease in the immunostaining for the KGDHC correlated roughly with the severity of degeneration. The KGDHC is more vulnerable to degeneration than complex II, III, and IV as noted in our previous immunohistochemical study. Even if secondary, the loss may play a role in the progression of the disease.

We isolated cDNA for dihydrolipoamide succinyltransferase from a human fibroblast cDNA library in lambda gt11. The cDNA revealed that the human dihydrolipoamide succinyltransferase lacked a sequence motif of an E3 and/or E1 binding site. This suggests that the human dihydrolipoamide succinyltransferase possesses a unique structure consisting of two domains in contrast with the dihydrolipoamide acyltransferases of other alpha-keto acid dehydrogenase complexes. In addition, we found that the human dihydrolipoamide succinyltransferase gene is located on chromosome 14 at q24.2-q24.3 and that a sequence related to the dihydrolipoamide succinyltransferase gene is located on chromosome 1 at p31. Interestingly, the gene for the dihydrolipoamide acyltransferase of the branched chain alpha-keto acid dehydrogenase complex is also located on chromosome 1p31 (Zneimer et al. (1991) Genomics 10, 740-747).

Physarum vitronectin-like protein with a molecular mass of 70 kDa cross-reacts with anti-bovine vitronectin and promotes cell-spreading (Miyazaki, K. et al. 1992. Exp. Cell Res., 199: 106-110.). The amino-terminal sequence of Physarum vitronectin-like protein is, however, distinct from those of animal vitronectins but shows significant sequence homology with dihydrolipoamide acetyltransferase, a component of pyruvate dehydrogenase complex. We have investigated the structural relationships between Physarum vitronectin-like protein and dihydrolipoamide acetyltransferase by using both antibody and protein-chemical methods. The vitronectin-like protein reacted with both anti-bovine vitronectin IgG and anti-rat pyruvate dehydrogenase complex IgG, indicating that it shares common antigenic determinant(s) with rat pyruvate dehydrogenase complex. Furthermore, sequencing studies of peptides obtained by lysylendopeptidase digestion indicated that internal sequences of Physarum vitronectin-like protein show significant homology with dihydrolipoamide acetyltransferase, but do not show any homology with the primary structures of authentic vitronectins. Immunocytochemistry revealed that the protein is widely localized in cytoplasm and nuclei of Physarum polycephalum, but is not present in the central area of vacuoles. Our results indicate that Physarum vitronectin-like protein is a molecule structurally and immunologically related to dihydrolipoamide acetyltransferase but functionally similar to animal vitronectin, although its localization is unique.

A complementary DNA (cDNA) clone of dihydrolipoamide acetyltransferase (E2) of the rat pyruvate dehydrogenase complex (PDC) was isolated from a lambda gt11 rat heart cDNA library. The amino acid sequence of a full mature protein of rat PDC-E2 was predicted by combination of the cDNA nucleotide sequence and the N-terminal amino acid sequence determined chemically. The amino acid sequence of rat PDC-E2 was well consistent with those of the E2 components of other alpha-ketoacid dehydrogenase complexes. These E2 components possess the sequence G-X-G-X-X-G, which is the consensus sequence for nucleotide binding sites of nucleotide binding proteins, in the E3 and/or E1 binding domains. The E2 components of the three alpha-ketoacid dehydrogenase complexes are suggested to be classified into three clusters separated during evolution.

The pyruvate dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex are multienzyme complexes consisting of three different enzymes. No significant similarity has been reported among the dehydrogenases which are component enzymes of these complexes, despite the presence of homology among the other component enzymes. Here we isolated cDNAs for the alpha and beta subunits of rat pyruvate dehydrogenase and they exhibited a significant similarity of the amino acid sequences among rat pyruvate dehydrogenase, 2-oxoisovalerate dehydrogenase (which is a dehydrogenase component of branched chain alpha-ketoacid dehydrogenase complex) and alpha-ketoglutarate dehydrogenase, suggesting that they have been derived from a common ancestral dehydrogenase. Our results suggested that the alpha and beta subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the alpha-ketoglutarate dehydrogenase. However, we could not find significant homology between rat pyruvate dehydrogenase and Gram-negative bacterial pyruvate dehydrogenase.

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

Pyruvate dehydrogenase complex and lipoamide dehydrogenase were purified from muscle of Ascaris lumbricoides var. suum which contains relatively a large amount of the complex. Molecular weights of three constituent enzymes of Ascaris pyruvate dehydrogenase complex were as follows; alpha- and beta-subunits of pyruvate dehydrogenase were 42,000 and 37,000, respectively, lipoate acetyltransferase was 76,000 and lipoamide dehydrogenase was 56,000. Furthermore, two unknown polypeptides having molecular weight of 46,000 and 41,000 were detected. Anti-Ascaris lipoamide dehydrogenase antibody precipitated three constituent enzymes and two unknown polypeptides, suggesting that lipoamide dehydrogenase not only binds tightly to complex, but also two unknown polypeptides bind tightly to complex.

Lipoamide dehydrogenases from various sources were purified and their immunochemical properties were compared. Antibody against rat lipoamide dehydrogenase reacted with rat, human, pig, pigeon and frog enzymes, but not with enzymes from E. coli, yeast and Ascaris. Anti-Ascaris enzyme and anti-E. coli enzyme antibodies reacted with Ascaris and E. coli enzymes, respectively. The pyruvate dehydrogenase subcomplex, which consists of pyruvate dehydrogenase and lipoate acetyltransferase, was prepared by releasing the lipoamide dehydrogenase from rat heart pyruvate dehydrogenase complex by anti-lipoamide dehydrogenase antibody. Lipoamide dehydrogenases from various sources were added to rat pyruvate dehydrogenase subcomplex and the complex overall activity was measured. Each lipoamide dehydrogenase effectively recovered the overall activity of rat pyruvate dehydrogenase subcomplex to 80% of the original activity.

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.

IgG-maleimide peroxidase, Fab'-maleimide peroxidase, polymer and monomer types of Fab'-periodate peroxidase were prepared from an antibody against rat lipoamide dehydrogenase, a component of the pyruvate dehydrogenase complex which is located in mitochondria. They were examined for immunohistochemical staining of the rat kidney. Fab'-maleimide peroxidase was the best for staining mitochondrial protein. IgG-maleimide peroxidase and the monomer type of Fab'-periodate peroxidase had the same intensity of staining. The polymer type of Fab'-periodate peroxidase could not stain the lipoamide dehydrogenase.

Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-tenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S]methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase.

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector lambda gt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified tha cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.

The effect of the antispasmodic agent, SA-504 subtle granules, was studied. Ninety mg per day of timepidium bromide subtle granules was given orally to 17 patients with renal or ureteral calculi for no less than fourteen days. SA-504 subtle granules as well as the capsule showed good antispasmodic effect and stimulated spontaneous passage of ureteral calculi. No subjective or objective side effects were observed.

Pyruvate dehydrogenase complex was purified from rat heart. A new component(mol.wt; 52,000) was found in the purified complex in addition to well known three component enzymes. This component(referred to as component X) was acetylated with [2-14C] pyruvate in the absence of CoA as well as lipoate acetyltransferase. The anti-lipoate acetyltransferase antibody reacted with component X and lipoate acetyltransferase, suggesting that component X shows homology with lipoate acetyltransferase in protein structure. cDNA for lipoate acetyltransferase was isolated from rat liver cDNA library in lambda gt 11. cDNA for lipoate acetyltransferase recognized two kinds of mRNAs of 3.5 Kb and 2.5 Kb.

The partial amino acid sequence of rat lipoate acetyltransferase was determined using the intact protein and the peptides derived from a digest with Achromobacter protease I. The results showed the amino-terminal sequence of the mature enzyme to be (N) Ser-Leu-Pro-Pro-His-Gln-Lys-Val-Pro-Leu-Pro-Ser- Leu-Ser-Pro-Thr-Met-Gln-Ala-Gly-Thr-Ile-Ala-Arg-Trp-Glu-Lys. In addition, the sequences of two possible lipoyl-binding sites in the subunit, which are very similar to each other, were established.

A b-type cytochrome and NADH-ferricyanide (FC) reductase were solubilized from Ascaris muscle microsomes by detergents and purified by column chromatography. The purified b-type cytochrome displayed absorption bands at 560 (alpha-peak), 525 (beta-peak), and 424 nm (gamma-peak), with a marked shoulder at 555 nm in the reduced from, 415 nm (gamma-peak) in the oxidized form. This absorption spectrum was different from that of rat liver microsomal cytochrome b5. The molecular weight was estimated to be about 100,000 by SDS-polyacrylamide gel electrophoresis, and the absorption spectrum of alkaline pyridine ferrohemochrome suggested that the prosthetic group of this cytochrome is protoheme. The molecular weight of the purified NADH-FC reductase was estimated to be about 55,000 by SDS-polyacrylamide gel electrophoresis. The purified reductase required NADH as a specific electron donor. The reductase efficiently reduced some redox dyes with NADH, but the reduction of cytochrome c was much slower. The purified reductase, like the membrane-bound reductase, was not inhibited by thiol reagents.

Testicular microlithiasis was found in a 30-year-old infertile man. The literature is reviewed and the possible influence of testicular microlithiasis on male infertility is discussed.

Two subcellular fraction, P-1 and P-2, were isolated by differential centrifugation from 0.25 M sucrose muscle homogenates of the parasitic roundworm, Ascaris lumbricoides suum. Morphological studies indicated that P-1 fraction consisted of intact mitochondria, whereas P-2 fraction consisted almost exclusively of vesicular components. The difference spectrum of Ascaris microsomes showed a characteristic b-type cytochrome spectrum with three distinct absorption peaks at 560, 525, and 424 nm. However, the alpha-peak at 560 nm was asymmetric with a shoulder at 555 nm. This microsomal b-type cytochrome was reduced by NADH, which was inhibited by rotenone and HgCl2. The reduced b-type cytochrome was easily reoxidized by shaking. NADH-oxidase activity observed in Ascaris microsomes was inhibited by rotenone, but not by KCN, NaN3, and antimycin A. On the other hand, NADH-cytochrome c and NADH-neotetrazolium (NT) reductase activities in Ascaris microsomes were not inhibited by antimycin A and rotenone, but were inhibited by HgCl2. Further observations indicated that neither HgCl2 nor rotenone inhibited Ascaris microsomal NADH-ferricyanide (FC) reductase activity, but rabbit antibody prepared against the purified NADH-FC reductase inhibited the NADH-cytochrome c reductase activity, the reduction of b-type cytochrome and the NADH-oxidase activity, as well as microsomal NADH-FC reductase activity.

Hemin degradation was observed in the presence of NADH and lipoamide dehydrogenase at pH 6.5-9.0. The degradation was inhibited by 80% by catalase and by 70% by superoxide dismutase. This system oxidized oxyhemoglobin to methemoglobin but only slightly degraded heme in hemoglobin.