Evaluation of the adequacy of oxygen consumption by tissues using the "anionic burst" parameter

A cDNA encoding farnesyl diphosphate (FPP) synthase (FPPS) has been cloned from a cDNA library of Artemisia annua. The sequence analysis showed that the cDNA encoded a protein of 343 amino acid (aa) residues with a calculated molecular weight of 39420 kDa. The deduced aa sequence of the cDNA was highly similar to FPPS from other plants, yeast and mammals, and contained the two conserved domains found in polyprenyl synthases including FPPS, geranylgeranyl diphosphate synthases and hexaprenyl diphosphate synthases. The expression of the cDNA in Escherichia coli showed enzyme activity for FPPS in vitro.     

Glycerol-3-phosphate acyltransferase in plants

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the transfer of an acyl group from an acyl donor to the sn-1 position of glycerol 3-phosphate. The plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm, respectively. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor. cDNAs for GPAT of chloroplasts have been cloned from several plants, and the gene for the enzyme has been cloned from Arabidopsis thaliana. The amino acid sequences deduced from the nucleotide sequences of cDNAs indicate that the product of translation is a precursor of about 460 amino acid residues, which consists of a leader sequence of about 70 amino acid residues and a mature protein of about 400 residues, with a molecular mass of about 42 kDa. Genetic engineering of the unsaturation of fatty acids has been achieved by manipulation of the cDNA for the GPAT found in chloroplasts and has allowed modification of the ability of tobacco to tolerate chilling temperatures.     

Mortality prediction in head trauma patients: performance of Glasgow Coma Score and general severity systems

A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 micromol x min(-1) x mg(-1). The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.     

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.     

The recombinant alpha isoform of protein kinase CK1 from Xenopus laevis can phosphorylate tyrosine in synthetic substrates

The cDNA coding for protein kinase CK1 alpha has been cloned from a Xenopus laevis cDNA library. The derived amino acid sequence of the protein contains 337 amino acids and has a calculated molecular mass of 38874 Da. The sequence is identical to that of the human CK1 alpha and to the bovine CK1 alpha, except that it is 12 amino acids longer than the latter protein. Southern blotting with a 264-bp probe demonstrates that four or more fragments are obtained upon digestion of genomic DNA with EcoR1 and Hind3, suggesting that X. laevis possesses a family of related CK1 genes. CK1 alpha was expressed in Escherichia coli as a glutathione transferase fusion protein (GT-CK1 alpha) and certain of its characteristics were determined. The recombinant GT-CK1 alpha fusion protein was found to have apparent Km values for ATP (12 microM), casein (1.5 mg/ml) and the specific peptide substrate RRKDLHDDEEDEAMSITA (180 microM) which are similar to those of the rat liver CK1 enzyme. The recombinant CK1 alpha activity is weakly inhibited by heparin, but strongly inhibited by poly(Glu80:Tyr20). This inhibition is competitive and shows an approximate K1 of 5 microM. CK1 alpha can phosphorylate the tyrosine residues of poly(Glu80:Tyr20) and the tyrosine residue in the synthetic peptide RRREEEYEEEE. This kinase preparation also autophosphorylates in serine, threonine and weakly in tyrosine.     

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.     

Molecular cloning of capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus (Korean viper)

The gene of capillary permeability-increasing enzyme-2 (CPI enzyme-2) was cloned from the cDNA library of Agkistrodon caliginosus and its nucleotide sequence was determined. Its sequence indicates that CPI enzyme-2 is synthesized as a pre-zymogen of 258 amino acid residues, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acids. The amino terminal sequence deduced from the cDNA sequence was exactly consistent with that of CPI enzyme-2 except for the substitution of an amino acid (Gly27-->Ser). The open reading frame is very similar to those of plasminogen activator and thrombin-like proteases cloned from other snakes. The clone encoding CPI enzyme-2 belongs to the serine protease family. The active site of the enzyme is highly conserved at His41, Asp86 and Ser180. Its possible glycosylation sites, Asn-X-Thr/Ser, are located at amino acid residues 20-22, 55-57, 79-81 and 97-99.     

Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP-AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST-clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide-derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77187 containing a C-terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP-AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP-AT.     

Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.     

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.     

The reductase domain of the human inducible nitric oxide synthase is fully active in the absence of bound calmodulin

Adenosine kinase (adk) from the moss Physcomitrella patens (Hedw.) B.S.G. was cloned from a cDNA library by functional complementation of an Escherichia coli purine auxotrophic strain. The length of the entire cDNA clone was 1175 bp with an open reading frame coding for a protein with a predicted molecular weight of 37.3 kDa. Southern analysis indicated the presence of a single adenosine kinase gene within the Physcomitrella genome. The deduced amino acid sequence had a 52% identity with the human adenosine kinase. The transfer of phosphate from ATP to adenosine resulting in AMP, as well as the phosphorylation of the cytokinin, isopentenyladenosine, to isopentenyladenosine monophosphate, was shown by in vitro enzyme assays using crude extracts from E. coli mutants expressing the adk cDNA clone and from Physcomitrella chloronemal tissue. Results from in vivo feeding of chloronemal tissue with tritiated isopentenyladenosine suggest that adenosine kinase plays an important role in the conversion of cytokinins towards their nucleotides in Physcomitrella.     

Cloning and high-level expression of alpha-galactosidase cDNA from Penicillium purpurogenum

The cDNA coding for Penicillium purpurogenum alpha-galactosidase (alphaGal) was cloned and sequenced. The deduced amino acid sequence of the alpha-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic alphaGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was to Trichoderma reesei AGLI. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.     

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.     

Sequence analysis of porcine gut GLI-1

A protein from porcine gut with 100 amino acid residues (porcine gut GLI-1) and having glucagon-like immunoreactivity has been characterized by partial sequences. The sequence of the C-terminal amino acid residues is -Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala and includes the C-terminal amino acid residue sequence (-Met-Asn-Thr) of porcine glucagon. Evidence is presented that the glucagon sequence -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- is found in the gut GLI-1 as well. The data support the theory that gut GLI-1 contains the full glucagon sequence and that gut GLI-1 and glucagon are formed from a common precursor.     

Molecular cloning and nucleotide sequence of the protyrosinase gene, melO, from Aspergillus oryzae and expression of the gene in yeast cells

The coding region of the protyrosinase gene, melO, from Aspergillus oryzae occupies 1671 base pairs of the genomic DNA and is separated into two exons by one intron. The full-length cDNA of the melO gene was cloned. Analysis of the 1617 base pairs nucleotide sequence revealed a single open reading frame coding 539 amino acid residues. The cDNA has been expressed in yeast cells. The predicted protein product derived from the melO gene is identified by Western blotting and activity determination. The predicted amino acid sequence of the gene product was compared with that of Neurospora crassa tyrosinase. A coupled pair of three histidine residues in the tyrosinase was assumed to correspond to Cu(II) ligands in the homologous tyrosinases from Streptomyces glaucescens and Homo sapiens.     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

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.     

Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase

A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite. IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides. Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3. Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases. The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases. The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.     

Cloning and expression of the programmed cell death regulator Bad in the rat brain

The Bcl-2 family of proteins consists of both antagonists (e.g. Bcl-2) and agonists (e.g. Bax) that regulate apoptosis and compete through dimerization. In the present study we cloned the cDNA encoding the rat brain BAD, a distant member of the Bcl-2 family that was shown to promote cell death. The cloned cDNA encoded a protein of 205 amino acids, containing three putative Bcl-2 homology domains (BH1, BH2 and BH3) and no C-terminal signal-anchor sequence. The predicted amino acid sequence was identical to the Bad-cDNA recently cloned from the rat ovary with the exception of a stretch of six amino acids, thus indicating the existence of two Bad alternative splice variants or a sequence artifact in the rat ovary Bad-cDNA. Immunohistochemical analysis in the rat brain revealed the exclusive expression of Bad in the epithelial cells of the choroid plexus, a result which is consistent with a very specialized function of Bad in the brain.