Purification, cDNA cloning and expression of human NG,NG-dimethylarginine dimethylaminohydrolase

cDNA encoding N(G),N(G)-dimethylarginine dimethylaminohydrolase from rat kidney had been cloned [Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N. & Ogawa, T. (1997) Biochim. Biophys. Acta 1337, 6-10]. The enzyme hydrolyzes N(G),N(G)-dimethyl-L-arginine and N(G)-monomethyl-L-arginine, which are known as endogenous inhibitors for the nitric oxide-generating system. In the present study, human N(G),N(G)-dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized. The cDNA clone encoding human N(G),N(G)-dimethylarginine dimethylaminohydrolase was isolated from a human kidney lambda gt10 library using a probe prepared from a plasmid containing the entire coding region of rat N(G),N(G)-dimethylarginine dimethylaminohydrolase. Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31,121 Da. The deduced amino acid sequence exhibits 93% identity with that of rat. The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme.     

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% alpha-helix and 9% beta-sheet were found.     

Partial structure of the gene encoding the 78 kDa gastrin binding protein excludes a close relationship with the peroxisomal trifunctional enzyme

A 10.9 kb porcine genomic clone encoding nucleotides 124-732 of the cDNA for the porcine 78 kDa gastrin-binding protein has been isolated and characterized. The coding sequence is interrupted by 7 introns, which vary in length from 93 to 3000bp. The positions of the intron/exon junctions are different from the junctions in the gene encoding the rat peroxisomal trifunctional enzyme. Despite 33% amino acid sequence identity between the two proteins it is concluded that the porcine gastrin binding protein is not closely related to the rat trifunctional enzyme.     

Molecular cloning and expression of amadoriase isoenzyme (fructosyl amine:oxygen oxidoreductase, EC 1.5.3) from Aspergillus fumigatus

Amadoriase is an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2. We previously reported the purification and characterization of two amadoriase isozymes from Aspergillus sp. that degrade both glycated low molecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V. M. (1997) J. Biol. Chem. 272, 3437-3443). To identify the primary structure of the enzymes, we have prepared a cDNA library from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody. The primary structure of the enzyme deduced from the nucleotide sequence comprises 438 amino acid residues with a predicted molecular mass of 48,798 Da. The deduced primary structure exhibits the presence of an ADP-binding motif near the NH2 terminus. The identity of the amadoriase II cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern blotting analysis revealed that amadoriase II was induced by fructosyl propylamine in a dose-dependent manner.     

Biocompatibility of poly(etherurethane urea) containing dehydroepiandrosterone

Mammalian quinolinate phosphoribosyltransferase (QPRTase) (EC 2.4.2.19) is a key enzyme in catabolism of quinolinate, an intermediate in the tryptophan-nicotinamide adenine dinucleotide (NAD) pathway. Quinolinate acts as a most potent endogenous exitotoxin to neurons. Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders. As the first step to elucidate molecular basis underlying the quinolinate metabolism, the cDNA encoding human brain QPRTase was cloned and characterized. Utilizing partial amino acid sequences obtained from highly purified porcine kidney QPRTase, a human isolog was obtained from a human brain cDNA library. The cDNA encodes a open reading frame of 297 amino acids, and shares 30 to 40% identity with those of bacterial QPRTases. To confirm that the cDNA clone encodes human QPRTase, its functional expression was studied in a bacterial host. Introduction of the human cDNA into a QPRTase defective (nadC) E. coli strain brought about an abrupt increase in QPRTase activity and allowed the cells to grow in the absence of nicotinic acid. It is concluded that the cloned cDNA encodes human QPRTase which is functional beyond the phylogenic boundary.     

Sequence of the fifth largest subunit of RNA polymerase II from plants

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.     

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).     

Functional magnetic resonance imaging of median nerve stimulation in rats at 2.0 T

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.     

Molecular cloning and functional expression of a phorbol ester-inducible 8S-lipoxygenase from mouse skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.     

Disproportionating enzyme (4-alpha-glucanotransferase; EC 2.4.1.25) of potato. Purification, molecular cloning, and potential role in starch metabolism

Disproportionating enzyme (D-enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) has been purified to homogeneity from potato tubers and its activity characterized. The enzyme catalyzes the transfer of maltooligosaccharides from one 1,4-alpha-D-glucan molecule to another, or to glucose. Maltooligosaccharides are effective donor molecules, but short chain amylose and amylopectin may also function as donors. Enzyme activity is not affected by inorganic phosphate, 3-phosphoglycerate, or hexose phosphates. A cDNA clone encoding the enzyme was isolated using oligonucleotide probes derived from partial peptide sequences of the purified enzyme. The identity of the cDNA clone was confirmed by expression in Escherichia coli resulting in D-enzyme activity. The amino acid sequence deduced from the cDNA shows significant homology with a 4-alpha-glucanotransferase from Streptococcus. The deduced sequence indicates the presence of an amino-terminal plastid transit peptide of 52 amino acid residues and a mature polypeptide of 524 residues. D-enzyme mRNA is present in leaves, stems, roots, and stolons but is most abundant in developing and mature tubers. The amount of mRNA in leaves increases in response to light and to sucrose added to the medium. These results are discussed in terms of the function of D-enzyme in potato starch metabolism.     

The abundant 31-kilodalton banana pulp protein is homologous to class-III acidic chitinases

We have identified and characterized the abundant protein from the pulp of banana fruit (Musa acuminata cv. Grand Nain), and have isolated a cDNA clone encoding this protein. Comparison of the amino terminal sequence of the purified 31 kDa protein (P31) suggests that it is related to plant chitinases. Western analyses utilizing rabbit anti-P31 antiserum demonstrate that this protein is pulp-specific in banana. A full-length cDNA clone homologous to class III acidic chitinase genes has been isolated from a pulp cDNA library by differential screening. The identity of this clone as encoding P31 was verified by comparisons between the amino-terminal peptide sequence and the cDNA sequence and cross-hybridization of the translation product of the cDNA clone with P31 antiserum. Northern and western blot analyses of RNA and protein isolated from banana pulp at different stages of ripening indicate that the cDNA and protein are expressed at high levels in the pulp of unripe fruit, and that their abundance decreases as the fruit ripens. Based on its expression pattern and deduced amino acid sequence and composition, we hypothesize that the physiological role of P31 is not for plant protection, but as a storage protein in banana pulp.     

Expression of cytokine receptors in the placenta in term and preterm labour

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (C alpha) was isolated by screening a mesenteric lymph node cDNA library with a porcine C alpha exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian C alpha sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum C alpha sequence had a nucleotide sequence identity of 57.7% with porcine C alpha, 51% with mouse C alpha, 46.7% with dog C alpha and 45.9% with human C alpha2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Purification and cDNA cloning of maize Poly(ADP)-ribose polymerase

Poly(ADP)-ribose polymerase (PADPRP) has been purified to apparent homogeneity from suspension cultures of the maize (Zea mays) callus line. The purified enzyme is a single polypeptide of approximately 115 kD, which appears to dimerize through an S-S linkage. The catalytic properties of the maize enzyme are very similar to those of its animal counterpart. The amino acid sequences of three tryptic peptides were obtained by microsequencing. Antibodies raised against peptides from maize PADPRP cross-reacted specifically with the maize enzyme but not with the enzyme from human cells, and vice versa. We have also characterized a 3.45-kb expressed-sequence-tag clone that contains a full-length cDNA for maize PADPRP. An open reading frame of 2943 bp within this clone encodes a protein of 980 amino acids. The deduced amino acid sequence of the maize PADPRP shows 40% to 42% identity and about 50% similarity to the known vertebrate PADPRP sequences. All important features of the modular structure of the PADPRP molecule, such as two zinc fingers, a putative nuclear localization signal, the automodification domain, and the NAD+-binding domain, are conserved in the maize enzyme. Northern-blot analysis indicated that the cDNA probe hybridizes to a message of about 4 kb.     

Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase

cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.