Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.). (9/1258)

We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs.  (+info)

Quality and safety evaluation of genetically engineered rice with soybean glycinin: analyses of the grain composition and digestibility of glycinin in transgenic rice. (10/1258)

The composition of nutritionally and physiologically important molecules in transgenic rice with the soybean glycinin gene was determined and compared with that of a non-transgenic control. Except for the levels of protein, amino acids and moisture, no marked differences were found between the two kinds of rice. The protein content of the transgenic rice was about 20% higher than the control (control, 6.5 g/100 g; transgenic, 8.0 g/100 g) with a concomitantly lower moisture content. This increased protein content mainly resulted from the increased glycinin expressed in the transgenic rice, and the protein was susceptible to gastric and intestinal digestion juices. In parallel with the increased protein content, some important amino acids lacking in quantity in normal rice were replenished.  (+info)

Copper, zinc, and magnesium levels in non-insulin dependent diabetes mellitus. (11/1258)

A relationship has been reported between trace elements and diabetes mellitus. This study evaluated the role of such a relationship in 83 patients with non-insulin dependent diabetes mellitus (40 men and 43 women), with a mean duration of diabetes of 3.9 +/- 3.6 years. Patients with nephropathy were excluded. Thirty healthy non-diabetic subjects were studied for comparative analysis. Subjects were subdivided into obese and non-obese. Diabetic subjects were also subdivided into controlled and uncontrolled groups; control was based on fasting blood glucose and serum fructosamine levels. Plasma copper, zinc and magnesium levels were analysed using a GBC 902 double beam atomic absorption spectrophotometer. Plasma zinc and magnesium levels were comparable between diabetic and non-diabetic subjects, while copper levels were significantly elevated (p < 0.01) in diabetic patients. Age, sex, duration and control of diabetes did not influence copper, zinc, or magnesium concentrations. We conclude that zinc and magnesium levels are not altered in diabetes mellitus, but the increased copper levels found in diabetics in our study may merit further investigation of the relationship between copper and non-insulin dependent diabetes mellitus.  (+info)

Aminopeptidase B is structurally related to leukotriene-A4 hydrolase but is not a bifunctional enzyme with epoxide hydrolase activity. (12/1258)

Aminopeptidase B (Ap B; EC 3.4.11.6) is a zinc-binding protein that contains the consensus sequence HEXXHX18E (324-347), conserved among the M1 family of metallopeptidases. To determine if these putative zinc-binding residues (His324, His328 and Glu347) and the active-site Glu325 are essential for the enzyme activity, we replaced the histidines with tyrosines and the glutamic acid residues with alanines using site-directed mutagenesis. The cDNAs were expressed in Escherichia coli, and the resulting recombinant proteins, named H324Y, E325A, H328Y and E347A, were purified to apparent homogeneity. None of the expressed mutated proteins showed aminopeptidase activity. The E325A enzyme contained 1 mol of zinc per mol of protein, and the other three mutants, H324Y, H328Y and E347A, did not contain significant amounts of zinc, as determined by atomic absorption spectrometry. From sequence-homology searches, Ap B is known to be closely related to leukotriene (LT)-A4 hydrolase (EC 3.3.2.6). We examined human placental Ap B and recombinant rat Ap B, both of which had been purified previously [Fukasawa, Fukasawa, Kanai, Fujii and Harada (1996) J. Biol. Chem. 271, 30731-30735], to determine whether or not they had epoxide hydrolase activities. However, neither enzyme hydrolysed LTA4 into LTB4. We then replaced some amino acids in the domain of the rat enzyme similar to the LTA4-binding site of LTA4 hydrolase. However, these mutants, Y408F, N409S and NE409-410SS also did not possess any epoxide hydrolase activity. We concluded that Ap B is an M1-family zinc metallopeptidase without epoxide hydrolase activity.  (+info)

Molecular and enzymic properties of recombinant 1, 2-alpha-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells. (13/1258)

For the construction of an overexpression system of the intracellular 1,2-alpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1, 2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-alpha-d-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type 'M8A') fastest, and 'M6C' {Manalpha1-3[Manalpha1-2Manalpha1-3(Manalpha1-6) Manalpha1-6]Manbeta1- 4GlcNAcbeta1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1.cm-1. Contents of the secondary structure (alpha-helix, beta-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 degrees C by differential scanning calorimetry. The calorimetric enthalpy, DeltaHcal, of the enzyme was calculated as 13.3 kJ.kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.  (+info)

Delta3,5,7,Delta2,4,6-trienoyl-CoA isomerase, a novel enzyme that functions in the beta-oxidation of polyunsaturated fatty acids with conjugated double bonds. (14/1258)

The mitochondrial metabolism of unsaturated fatty acids with conjugated double bonds at odd-numbered positions, e.g. 9-cis, 11-trans-octadecadienoic acid, was investigated. These fatty acids are substrates of beta-oxidation in isolated rat liver mitochondria and hence are expected to yield 5,7-dienoyl-CoA intermediates. 5, 7-Decadienoyl-CoA was used to study the degradation of these intermediates. After introduction of a 2-trans-double bond by acyl-CoA dehydrogenase or acyl-CoA oxidase, the resultant 2,5, 7-decatrienoyl-CoA can either continue its pass through the beta-oxidation cycle or be converted by Delta3,Delta2-enoyl-CoA isomerase to 3,5,7-decatrienoyl-CoA. The latter compound was isomerized by a novel enzyme, named Delta3,5,7,Delta2,4, 6-trienoyl-CoA isomerase, to 2,4,6-decatrienoyl-CoA, which is a substrate of 2,4-dienoyl-CoA reductase (Wang, H.-Y. and Schulz, H. (1989) Biochem. J. 264, 47-52) and hence can be completely degraded via beta-oxidation. Delta3,5,7,Delta2,4,6-Trienoyl-CoA isomerase was purified from pig heart to apparent homogeneity and found to be a component enzyme of Delta3,5,Delta2,4-dienoyl-CoA isomerase. Although the direct beta-oxidation of 2,5,7-decatrienoyl-CoA seems to be the major pathway, the degradation via 2,4,6-trienoyl-CoA makes a significant contribution to the total beta-oxidation of this intermediate.  (+info)

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2. (15/1258)

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4 degrees c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k1) was 2.3 x 10(7) M-1 s-1 for PGHS-1 and 2.5 x 10(7) M-1 s-1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 10(2)-10(3) s-1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that the k2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.  (+info)

Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin. (16/1258)

In addition to recognizing and repairing mismatched bases in DNA, the mismatch repair (MMR) system also detects cisplatin DNA adducts and loss of MMR results in resistance to cisplatin. A comparison was made of the ability of MMR-proficient and -deficient cells to remove cisplatin adducts from their genome and to reactivate a transiently transfected plasmid that had previously been inactivated by cisplatin to express the firefly luciferase enzyme. MMR deficiency due to loss of hMLH1 function did not change the extent of platinum (Pt) accumulation or kinetics of removal from total cellular DNA. However, MMR-deficient cells, lacking either hMLH1 or hMSH2, generated twofold more luciferase activity from a cisplatin-damaged reporter plasmid than their MMR-proficient counterparts. Thus, detection of the cisplatin adducts by the MMR system reduced the efficiency of reactivation of the damaged luciferase gene compared to cells lacking this detector. The twofold reduction in reactivation efficiency was of the same order of magnitude as the difference in cisplatin sensitivity between the MMR-proficient and -deficient cells. We conclude that although MMR-proficient and -deficient cells remove Pt from their genome at equal rates, the loss of a functional MMR system facilitates the reactivation of a cisplatin-damaged reporter gene.  (+info)