Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.

Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase.

A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128,353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.

Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue.

Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site. Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine-238 to be the site of modification. Site-directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity. Lysine-238 seems to interact with the gamma-phosphate group of ATP but is not involved in catalysis.

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC.

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.

Comparative affinity labeling with reactive UDP-glucose analogues: possible locations of five lysyl residues around the substrate bound to potato tuber UDP-glucose pyrophosphorylase.

By using two reactive analogues of UDP-Glc, uridine di- and triphosphopyridoxals, we have recently probed the substrate-binding site in potato tuber UDP-Glc pyrophosphorylase [EC 2.7.7.9]. In this work, pyridoxal diphospho-alpha-D-glucose was used for the same purpose. This compound is also a reactive UDP-Glc analogue but having its reactive group on the opposite side of the pyrophosphate linkage to those of the above two compounds. The enzyme was rapidly inactivated when incubated with the compound at very low concentrations followed by reduction with sodium borohydride. The inactivation was almost completely prevented by UDP-Glc and UTP. Complete inactivation correspond to the incorporation of 1.0 mol of the reagent per mol of enzyme monomer. The label was found to be distributed in five lysyl residues (Lys-263, Lys-329, Lys-367, Lys-409, and Lys-40. All of these results were similar to those obtained previously with the other compounds, suggesting the presence of a cluster of five lysyl residues at or near the substrate-binding site of this enzyme. However, the incorporations of labels into each lysyl residue differed depending on the compounds used. The substrate retarded the incorporations in different manners. Based on the combined results of the present and previous studies, a hypothetical model is presented for the possible locations of the five lysyl residues around the substrate bound to the enzyme. This model is consistent with the kinetic properties of mutant enzymes in which the five lysyl residues were individually replaced by glutamine via site-directed mutagenesis.

Overproduction and characterization of recombinant UDP-glucose pyrophosphorylase from Escherichia coli K-12.

Using oligonucleotide probes synthesized on the basis of partial amino acid sequences, we have cloned and sequenced the gene of Escherichia coli K-12 encoding UDP-glucose pyrophosphorylase. The gene consists of 906 base pairs and encodes a polypeptide of 302 amino acid residues with a calculated molecular weight of 32,941. Its nucleotide sequence was found to be identical with that recently registered (EMBL, X59940) for a gene coding for an unknown 33-kDa protein, which was later annotated as UDP-glucose pyrophosphorylase on the basis of genetic studies. The UDP-glucose pyrophosphorylase gene, mapped at 27.3 min in the E. coli chromosome, complemented the galU mutation, which renders the bacterium unable to ferment galactose. The recombinant enzyme overproduced in E. coli cells and purified to homogeneity catalyzed the synthesis and pyrophosphorolysis of UDP-glucose by a sequential mechanism. The enzyme required Mg2+ for maximal activity and was inhibited by free UTP and pyrophosphate. The E. coli enzyme shows significant sequence similarities with the enzymes from Acetobacter xylinum and Salmonella typhimurium. However, little or no similarity was found with the eukaryotic enzymes that are involved in the biosynthesis of storage carbohydrates, or with other enzymes acting on similar sugar nucleotides. Thus, UDP-glucose pyrophosphorylases participating in diverse metabolic pathways can be classified structurally into the prokaryotic and eukaryotic groups, even though they have almost identical catalytic properties.

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing.

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.

Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrier protein as a substrate.

We developed a simple and sensitive method for assessing holocarboxylase synthetase (HCS) activity that is based on measuring incorporation of [3H]biotin into apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E. coli. Kinetic analysis of HCS from normal fibroblasts showed that the Km for biotin was 260 +/- 94 nmol/l (mean +/- S.D.; n = 5). In contrast, the Km values of HCS from two cell lines derived from patients with HCS deficiency were 7200 and 3700, clearly distinguishable from the control value. The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected. Our method is useful for enzymatic diagnosis of HCS deficiency and characterization of HCS.

Clinico-laboratory characteristics of patients with dermatomyositis accompanied by rapidly progressive interstitial lung disease.

The clinico-laboratory features of 16 patients with dermatomyositis (DM) were compared between patients with accompanying rapidly progressive interstitial lung disease (RP-ILD, n = 7) and those with chronic interstitial lung disease (C-ILD, n = 9), and also between deceased (seven RP-ILD and three C-ILD) and living patients (six C-ILD). The extent of muscle weakness of the extremities and frequency of autoantibody positivity were significantly lower in DM patients with RP-ILD than in DM patients with C-ILD. Furthermore, significantly lower serum creatine kinase/lactate dehydrogenase levels (0.26+/-0.27) were found in the 10 patients who died than in the six living patients (1.21+/-1.09). A higher CD4+/CD8+ T-lymphocyte ratio in the peripheral blood (3.51+/-2.65) was detected in the four DM patients with RP-ILD who died than in the six living DM patients with C-ILD (1.22+/-0.49).

The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs.

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be greater than or equal to 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.

A rapid microassay for dirofilarial antibodies by using antigen-coated nitrocellulose paper.

This study describes an enzyme (horseradish peroxidase)-linked immunosorbent assay using nitrocellulose (NC) paper (Dot-ELISA) for the detection of antibodies against Dirofilaria immitis in dogs. Dirofilarial antigens prepared were directly bound on NC paper set into a microfiltration apparatus to ensure their uniformity. Of the 23 infected dogs tested, 21 were determined positive by visual observation of brown-colored spots on NC paper. The positive and negative readings were further confirmed by using a densitometer. Sera from 15 non-infected and 17 other parasite-infected dogs were all negative, i.e. no false positive readings and low cross-reactivities were found using this technique. Also, a large number of samples could be assayed simultaneously within 1.5 h. The Dot-ELISA technique reported here may be useful to veterinarians and also may be a means of field surveying human filariasis.

Carbamazepine-induced systemic lupus erythematosus-like disease.

A 14-year-old female developed systemic lupus erythematosus (SLE)-like symptoms, rash, fever, leukopenia and positive anti-nuclear antibody (ANA) two weeks after administration of carbamazepine (CBZ; Tegretol) used against benign Rolandic epilepsy. Clinical symptoms and leukopenia normalized after discontinuation of CBZ and administration of prednisolone at 40 mg. The cases of CBZ-induced SLE reported in the literature were reviewed.

Primary antiphospholipid syndrome presenting with cerebral ischemia, thrombocytopenia, anemia and proteinuria successfully treated with warfarin potassium.

A 30-year-old woman with primary antiphospholipid syndrome (PAPS) presented with cerebral ischemia, thrombocytopenia, anemia and proteinuria. Administration of warfarin potassium, without concomitant corticosteroid administration, significantly improved all of these symptoms along with a decrease in the titers of antiCL-beta2-GP-I antibodies and a shortening of prolonged APTT. Therefore, the antiphospholipid antibodies in this patient could have been evoked by vitamin-K-dependent coagulation factors or plasma proteins which are assumed to undergo conformational changes exposing cryptic epitopes. This case report provides clues to the mechanisms underlying the production of antiphospholipid antibodies in patients with PAPS.

Two patients with polymyositis or dermatomyositis complicated with massive pleural effusion.

Two patients with polymyositis (PM) or dermatomyositis (DM) complicated with massive pleural effusion are reported here. Both patients presented a high-grade fever, pleural effusion prominent on the right, and good response to steroid therapy. In a 50-year-old woman with PM, combined process of pleural inflammation, cardiomyopathy and coexisting hypothyroidism were considered to be responsible for the accumulation of the massive pleural effusion. However, in a 34-year-old man with DM, pleural inflammation associated with interstitial pneumonia or pleural microvasculopathy in DM was considered to be responsible for the accumulation of the massive pleural effusion.

A case of autoimmune hemolytic anemia induced by IFN-beta therapy for type-C chronic hepatitis.

A 51-year old woman who had type-C chronic hepatitis developed autoimmune hemolytic anemia after 4 weeks treatment with a total 48 million interferon-beta, 6 million 8 times. Indirect Coomb's test turned from negative to positive through IFN therapy and a complication of asymptomatic primary biliary cirrhosis was confirmed by histologic findings of the biopsied liver which was performed during IFN therapy and a presence of anti-mitochondrial antibody. After stopping IFN therapy and adding prednisolone anemia improved in getting negative indirect Coombs test but remaining positive direct Coombs test.

Idiopathic thrombocytopenic purpura complicated by intracranial hemorrhage in a 78-year-old male.

A 78-year-old male was admitted to our hospital with cutaneous hemorrhage, aphasia and somnolence. On physical examination, he appeared drowsy and complained of epistaxis and multiple purpura. His platelet count was 8,000/microliters with a high level of PA-IgG, but the coagulation time was normal. A CT scan of the head revealed a left temporal and right frontal hemorrhage. Bone marrow aspiration revealed abundant megakaryocytes. He was diagnosed as having idiopathic thrombocytopenic purpura with intracranial hemorrhage. He was treated with prednisolone and azathioprine in addition to platelet infusions and high doses of gamma-globulin. After two months of treatment, the platelet count had risen to 40,000/microliters and his symptoms had subsided.