Kichiko Koike

Fluorescent analysis of α-keto acids in serum and urine by high-performance liquid chromatography

A procedure for rapid separation and microquantitative determination of various alpha-keto acids in serum and urine was developed. The procedure used reverse-phase high-performance liquid chromatography of the alpha-keto acids after derivatization into fluorescent quinoxalines by reaction with o-phenylenediamine. Deproteinization of serum with tungstic acid or methanol facilitated a constant recovery of alpha-keto acids. The useful range of analysis of seven alpha-keto acids by isocratic chromatography was from 10 to 250 pmol. The fluorescence emission was measured at 410 nm with excitation at 350 nm. The data obtained from samples of patients with chronic pyruvic acidemia and maple syrup urine disease, confirmed the usefulness of the method in clinical applications. A slightly modified procedure was needed for the analysis of oxaloacetic acid and phenylpyruvic acid.

Peripheral B lymphocyte repertoire to mitochondrial antigen in primary biliary cirrhosis-positive correlation between the disease activity and the frequency of circulating B lymphocytes specific for pyruvate dehydroge nase complex

B lymphocytes committed to the production of IgG antibodies (Abs) to mitochondrial antigen such as pyruvate dehydrogenase complex(PDC) were quantitated in the peripheral blood of patients with primary biliary cirrhosis(PBC) using Epstein-Barr virus as a polyclonal activator of human B lymphocytes. B lymphocytes committed to the production of IgG Abs to PDC were found in high frequency in patients with PBC(0.54 +/- 0.16%, mean value +/- SE, of total IgG-producing B lymphocytes) in contrast to type C chronic hepatitis and healthy subjects in which they were less than 0.01%. The frequency of these B lymphocytes specific for PDC increased in parallel to the progression of the Scheuers stage from I to II (stage I: 0.35 +/- 0.23%, stage II: 1.04 +/- 0.32%), but decreased with further progression to stage IV (stage III: 0.39 +/- 0.21%, stage IV: 0.07 +/- 0.06%). In addition, B lymphocytes specific for PDC decreased in the peripheral blood during the administration of cyclosporin A; this was accompanied by an improvement of lymphocyte infiltration severity in the liver. These data indicate that B lymphocytes specific for PDC are present in the peripheral blood of patients with PBC and their frequency reflects the degree of the lymphocyte infiltration in the liver.

Immunochemical detection of unrepaired cyclobutane-type pyrimidine dimers of DNAs extracted from human skin tumours.

SummaryUnrepaired cyclobutane-type pyrimidine dimers of DNA extracted from human skin tumours were examined by an immunoblotting method using polyclonal antibodies raised against UV-irradiated calf thymus DNA. A total of 40 DNA samples extracted from seven SCC lesions, two AK lesions, two lymphomas, one basal cell epithelioma, one eccrine poroma, one neurofibroma of Recklinghausens disease, on verruca vulgaris, four femoral normal skins and white blood cells of 21 humans were studied by immunoblotting using this antibody. Two of the 40 DNAs examined, one from facial actinic keratosis (AK) and one from a squamous cell carcinoma (SCC) which developed form facial AK formed immunoprecipitates. It was found, using photoreactivation enzyme plus visible light, that both immunoprecipitates were cyclobutane-type pyrimidine dimers. In addition, immunofluorescent studies on AK tissue were positive in an immunoblotting assay and revealed that the unremoved photodamage in DNA remained in the nucleus of AK cells. These findings indicate that these tumour cells may be deficient in the enzyme function for repairing photoproduct damage. The unrepaired cyclobutane-type pyrimidine dimer in AK cells might reflect the genetic process in multistage carcinogenesis as well as in xeroderma pigmentosum.

Crystallization and preliminary X-ray analysis of pig E3, lipoamide dehydrogenase

Pig heart lipoamide dehydrogenase was crystallized by the hanging-drop vapor-diffusion method. X-ray diffraction patterns show that the hexagonal crystals have unit-cell dimensions of a = b = 359. 3, c = 140.5 A. The crystal structure has been preliminarily solved by the molecular-replacement method in the space group P6322. Three dimeric molecules have been found in the asymmetric unit, one of them located on the crystallographic twofold axis.

Proteinase-catalyzed activation of porcine heart muscle pyruvate dehydrogenase and identification of its cleavage site

Porcine heart muscle pyruvate dehydrogenase (PDH, EC 1.2.4.1) with subunit composition alpha 2 beta 2 catalyzes the initial decarboxylation step of an oxidative decarboxylation sequence of pyruvate. Highly purified PDH, was further activated several-fold by limited digestion with trypsin, Staphylococcus aureus V8 proteinase (V8) or papain. The activation with these proteinases required about 10 min to attain a maximal level, lasted 1/2-2 h and thereafter decreased gradually. Addition of an inhibitor of each proteinase resulted in an immediate cessation of any further changes in the enzymatic activity. The optimal pH of the proteinase-activated PDH was not affected. Proteinases increased the maximum velocity and the apparent Km values for pyruvate, but the Hill coefficients for pyruvate were unchanged. Proteinase-activated PDH was capable of associating two other component enzymes to produce large unit resembling the native complex. The Coomassie brilliant blue stained gels after SDS-PAGE showed that the PDH alpha subunit (41 kDa) was cleaved by trypsin or V8 into two major fragments (31 and 10 kDa), whereas PDH beta was unaffected. By amino-terminal sequence analyses of these fragments the trypsin cleavage sites were identified as Arg-273 and Arg-282 and the V8 cleavage sites were Glu-277 and Glu-280.

Hepatic phosphoenolpyruvate carboxykinase deficiency: a neonatal case with reduced activity of pyruvate carboxylase

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is a rate-limiting enzyme of the gluconeogenic pathway. In two of six reported cases, reduced activity of pyruvate carboxylase (EC 6.4.1.1) has been reported, but this is not well established. We present here a further case of phosphoenolpyruvate carboxykinase deficiency with reduced activity of pyruvate carboxylase

Molecular Cloning of cDNAs for α and β Subunits of Human Pyruvate Dehydrogenasea

The cDNAs encoding human PDH alpha and PDH beta were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening, followed by colony hybridization from a human foreskin fibroblast cDNA library. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH alpha and one of 1.69 kb for PDH beta, respectively, allowing us to predict the complete amino acid sequences of the precursor and mature proteins of these two subunits. The amino acid sequences of the amino-terminal regions of the two subunits of human PDH were highly homologous with those of mature porcine PDH. The amino acid sequences of phosphorylation sites determined in PDH alpha of the bovine and porcine enzymes were also conserved in the human PDH alpha. Blot analysis of HeLa cell poly(A)+ RNA and the transcriptional product of the two cDNAs showed a single mRNA of 1.8 kb for PDH alpha and one of 1.7 kb for PDH beta. The precursor proteins of PDH alpha and PDH beta were detected by immunoprecipitation from an 35S-labeled, cell-free translation system. Our sequence of PDH alpha cDNA was compared with those of two other origins. The differences among these three PDH alpha cDNAs have been discussed.

Crystallization and preliminary X-ray analysis of the full-size cubic core of pig 2-oxoglutarate dehydrogenase complex

The full-length (untruncated) dihydrolipoamide succinyltransferase from pig heart was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction patterns indicate that the crystal belongs to space group I432, with unit-cell parameter a = 189.9 A. The crystal structure has been preliminarily solved at 7 A resolution by the molecular-replacement method. The unit cell contains two cubic cores, in each of which 24 subunits of E2 are associated according to crystallographic 432 symmetry. At the corners of each cubic core, the catalytic domains of E2s form a trimer through tight interactions around the crystallographic threefold axes. In the electron-density maps, many small broad peaks are observed in regions expected to contain the remaining N-terminal domains (the E1/E3-binding domain and the lipoyl domain), suggesting flexibility of these domains relative to the core. The architecture of the cubic core is similar to that of the other truncated E2s. In the unit cell, however, the core-core contact occurs in a different direction from that found for the truncated proteins.

Chronic Acidemia Due to a Pyruvate Dehydrogenase Deficiency in the Pyruvate Dehydrogenase Complex, with Evidence of Abnormalities of the α and β Subunits of the Enzymea

It has been reported that a genetic pyrpvate dehydrogenase (PDH) deficiency in infants results in progressive neurological disease with persistent lactic and pyruvic acidemia and growth retardation. In Japan, among more than 400 patients with increased blood levels of pyruvate and lactate, we have diagnosed over the past ten years eight patients in whom there is substantial biochemical evidence for abnormalities of the PDH component of the PDH multienzyme complex. This work was based on our development of highly sensitive enzyme assay procedures closely dependent on pyruvate metabolism and by analytical procedures for a-keto acids in blood and urine.’ These studies were aimed a t providing information concerning the nature of genetic mutants leading to molecular diseases (PDH deficiency) and a t aiding in carrier detection, prenatal diagnosis, and treatment. The eight cases of PDH deficiency were further characterized by immunoblotting of the two subunit proteins (PDHa and PDHP) with specific antisera directed to the two porcine heart subunits. As shown in FIGURE 1, anti-porcine PDHa serum highly cross-reacted not only with porcine PDHa (A, lanes 6 and 7), but also with human liver PDHa (A, lane 1); and, similarly, anti-porcine PDHp serum cross-reacted with both porcine PDHp (B, lanes 6 and 7) and human PDHp (B, lane 1). In the immunoblots of the liver extract from patient M, the PDHa and PDHp bands were absent, in contrast to the other two components of PDH complex, and new, larger bands appeared. In liver extract from patient S, no bands were detected; however, in extracts from patients H and T, two normal size bands were detected. These results strongly suggested that impairments in the PDH complex of patients M and S are due to absence or abnormalities of the PDH component and that further studies of PDH on the gene level are indispensable. Recently we have succeeded in the cloning and sequencing of human PDHa and PDHp cDNAs.’ Preliminary studies of the genomic structure of PDHa and PDHp by Southern blot analysis using the two nick-translated radiolabeled cDNAs as probes have revealed simple digested fragment patterns with restriction enzymes, as shown in