Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.     

Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin.

Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin. These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin. Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities. The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene. Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not. Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity. Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule. Wild-type toxin inactivated by EDTA contained two zinc atoms. These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136. The variant toxin at H-148 possessed only one zinc atom. Wild-type toxin and the variant toxin at H-148 showed [65Zn]2+ binding, but the variant toxins at H-68, -126, and -136 did not. Furthermore, [65Zn]2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+. These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.     

Subunit composition of RNA polymerase II from the fission yeast Schizosaccharomyces pombe

The subunit composition of RNA polymerase II (polII) was compared between the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. For this purpose, we partially purified the enzyme from S. pombe. Judging from the co-elution profiles in column chromatographies of both the RNA polymerase activity and the two large subunit polypeptides (subunit 1 (prokaryotic β' homologue) and subunit 2 (β homologue)), the minimum number of S. pombe polII-associated polypeptides was estimated to be ten, less than the proposed subunit number of the S. cerevisiae enzyme. These ten putative subunits of S. pombe polII correspond to subunits 1, 2, 3, 5, 6, 7, 8, 10, 11 and 12 of the S. cerevisiae counterparts     

Graphics of RNA secondary structure; towards an object-oriented algorithm

We present a new algorithm for the display of RNA secondarystructure. The principle of the algorithm is entirely differentfrom those currently in use in that our algorithm is ‘objectoriented’ while currrent algorithms are ‘procedural’.The circular RNA molecule of chrysanthemum stunt viroid wasused as input data for demonstrating the operation of the program.The major interest of this method will be found in its potentialuse in simulation graphics of RNA folding processes. Received on October 9, 1986; accepted on February 17, 1987     

Characterization of a new fetal hemoglobin variant, Hb F Izumi A gamma 6Glu replaced by Gly, by molecular secondary ion mass spectrometry

Molecular secondary ion mass spectrometry has characterized the structure of a new fetal hemoglobin variant, Hb F Izumi, without separation of peptides or amino acid analysis. First, the mass spectrum of a tryptic digest of the abnormal gamma globin revealed a decreased by 72 mass units in the molecular mass of peptide T-1,2, indicating the presence of a Glu leads to Gly substitution. Next, the analysis of the digest produced by the addition of staphylococcal protease, which specifically cleaves glutamyl peptide bonds, determined the site of substitution at 6th glutamic acid residue in peptide T-1,2 which contains two glutamic acid residues. Since this mass spectrometric approach provides digitalized data on peptide analysis, we call it 'digit printing'. The high sensitivity of this technique is especially promising for the analysis of molecular abnormality in various genetic disorders.     

Purification of ferredoxins and their reaction with purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Four ferredoxin (Fd) fractions, namely, FdA-D were purified from the green sulfur bacterium Chlorobium tepidum. Their absorption spectra are typical of 2[4Fe-4S] cluster type Fds with peaks at about 385 and 280 nm and a shoulder at about 305 nm. The A(385)/A(280) ratios of the purified Fds were 0.76-0.80. Analysis of the N-terminal amino acid sequences of these Fds (15-25 residues) revealed that those of FdA and FdB completely agree with those deduced from the genes, fdx3 and fdx2, respectively, found in this bacterium (Chung and Bryant, personal communication). The N-terminal amino acid sequences of FdC and FdD (15 residues) were identical, and agree with that deduced from the gene fdx1 (Chung and Bryant, personal communication). The A(385) values of these Fds were unchanged when they were stored for a month at -80 degrees C under aerobic conditions and decreased by 10-15% when they were stored for 6 days at 4 degrees C under aerobic conditions, indicating that they are not extremely unstable. In the presence of Fd-NADP(+) reductase from spinach, and a purified reaction center (RC) preparation from C. tepidum composed of five kinds of polypeptides, these Fds supported the photoreduction of NADP(+) at room temperature with the following K(m) and V(max) (in micromol NADP(+) micromol BChl a(-1) h(-1)): FdA, 2.0 microm and 258; FdB, 0.49 microM and 304; FdC, 1.13 microM and 226; FdD, 0.5 microM and 242; spinach Fd, 0.54 microM and 183. The V(max) value of FdB was more than twice that previously reported for purified RC preparations from green sulfur bacteria.     

Critical evaluation of α1- and β2-microglobulins in urine as markers of cadmium-induced tubular dysfunction

The purpose of the study was to examine the validity of alpha1-microglobulin (alpha1-MG) in comparison with popularly used beta2-microglobulin (beta2-MG). A database was revisited to select ca. 7,500 spot urine samples (of adequate urine density) from non-pregnant, non-lactating and never-smoking adult women. The validity of the MGs was examined in terms of stability of the MG-uria prevalence in urine samples of various creatinine (CR or cr) concentration or specific gravity (SG or sg). Comparisons were made for MGs as observed (e.g., alpha1-MGob), as corrected for CR (e.g., alpha1-MGcr) and as corrected for SG of 1.016 (e.g., alpha1-MGsg). A cut-off value of 5.7 mg/g cr (or mg/l) for alpha1-MG was deduced from a cut-off value of 400 microg/g cr (or mcirog/l) for beta2-MG, because the correlation between alpha1-MGcr and beta2-MGcr was statistically significant. The prevalence of a 1-MGsg-uria was essentially unchanged (i.e., from a low of 13.6% to a high of 17.0%, or 1.2 times) except for in very dense or very thin urine samples, in contrast, beta2-MGcr-uria showed a substantial increase (from 0.0% to 2.8% with an infinite rate) as a reverse function of a decrease in CR in urine. The prevalence of uncorrected markers, i.e., alpha1-MGob-uria and beta2-MGob-uria, showed even greater CR- or SG-dependent changes. Thus, it appeared prudent to consider a alpha-MGsg rather than beta2-MGcr as a marker of tubular dysfunction among a general population with various urine density.     

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.     

Type III Cu mutants of Myrothecium verrucaria bilirubin oxidase

Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.