Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Further studies on aspartate aminotransferase of thermophilic methanogens by analysis of general properties, bound cofactors, and subunit structures.

Aspartate aminotransferase (AspAT) [EC 2.6.1.1] of thermophilic methanogen was further characterized with the enzyme from Methanobacterium thermoautotrophicum strain FTF-INRA as well as M. thermoformicicum strain SF-4. AspAT of strain FTF-INRA was similar in the amino donor specificity to the enzyme of M. thermoformicicum strain SF-4, in that it was active on L-cysteine and L-cysteine sulfinate in addition to L-glutamate and L-aspartate. The enzymes gave similar absorption spectra having maxima at around 326 and 415 nm with no pH-dependent shift but were found to contain 1 mol of tightly bound pyridoxal 5'-phosphate (PLP) per subunit. Reconstitution of each apoenzyme with added PLP resulted in partial recovery of the original enzymatic activity, suggesting a significant conformational change of the active site region upon removal of the cofactor. Polyacrylamide gel electrophoresis (PAGE) and gel filtration analyses revealed a tetrameric structure (180 kDa) of identical subunits with a molecular mass of 43 kDa for each of these enzymes. Electric current was found to affect the interaction or affinity of each subunit, promoting dissociation of the native enzyme into the monomeric form. Alkaline treatment was effective only for dissociation of the enzyme from strain SF-4. They were distinguishable by the more rapid reassociation of the monomer to the native aggregated form in the enzyme of strain FTF-INRA.     

Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The primary structure of the Cytisus scoparius seed lectin and a carbohydrate-binding peptide.

The complete amino acid sequence of 2-acetamido-2-deoxy-D-galactose-binding Cytisus scoparius seed lectin II (CSII) was determined using a protein sequencer. After digestion of CSII with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSII with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid residues of concanavalin A (Con A) involved in the metal binding site are highly conserved among those of CSII. A carbohydrate-binding peptide of CSII was obtained from the endoproteinase Asp-N digest of CSII by affinity chromatography on a column of GalNAc-Gel. This peptide was retained on the GalNAc-Gel column and was presumed to have affinity for the column. The amino acid sequence of the retarded peptide was determined using a protein sequencer. The retarded peptide was found to correspond to the putative metal-binding region of Con A. These results strongly suggest that this peptide represents the carbohydrate-binding and metal ion-binding sites of CSII.     

Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN [correction of INF] and augment IFN-mediated [correction of INF] natural killer activity.

Thirty-mer single-stranded oligonucleotides, with a sequence chosen from the known cDNA encoding the 64-kDa protein named Ag A or the MPB-70 protein of Mycobacterium bovis BCG and the human cellular proteins such as complement component 1 inhibitor and Ig rearranged lambda-chain, were used to dissect the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. Three with the hexamer palindromic sequence as GACGTC were active, whereas two kinds of oligonucleotides with no palindrome were inactive. The oligonucleotides containing at least one of the different palindromic sequences showed no activity. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT, or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotides to augment NK cell activity. Stimulation of spleen cells with the substituted oligonucleotide, A4a-AAC, induced production of significant amounts of IFN-alpha/beta and small amounts of IFN-gamma. Augmentation of NK activity of the cells by the oligonucleotide was ascribed to IFN-alpha/beta production. These results strongly suggest that the presence of the unique palindromic sequences, such as GACGTC, AGCGCT, and AACGTT, but not ACCGGT, is essential for the immunostimulatory activity of oligonucleotides.     

Isolation and partial characterization of an 87-kilodalton beta-1,3-glucanase from Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces at least two extracellular beta-1,3-glucanases that lyse fungal cell walls. One of these extracellular enzymes was purified to homogeneity. The molecular mass was 87 kDa, and the pI was 4.3. The optimum temperature of the enzyme reaction was 70 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate. The pH range of the enzyme was broad (pH 4.5 to 9.0), and the optimum pH was 6.5. The enzyme is an endo beta-1,3-glucanase and has a random cleavage pattern.     

DNA ligase I mRNA and enzyme levels in human hematopoietic cells under dimethyl sulfoxide-induced growth-arrest and differentiation.

About half the activity level of DNA ligase I in cycling human lymphoblastoid cells (Raji and Akata) remained in the cells arrested at G1 by a 4-day treatment with 1.5% dimethyl sulfoxide (DMSO), and one-third the enzyme activity in actively growing promyelocytic leukemia cells HL-60 was detected in the terminally differentiated cells after DMSO-treatment. In contrast, DNA ligase I mRNA was negligible in the G1-arrested Raji and differentiated HL-60 cells. The steady-state mRNA level was increased 9 h after release from DMSO in the G1-arrested Raji cells and reached a maximum at 18 h. These results indicate that gene expression of human DNA ligase I, but not activity level of the enzyme, is closely correlated with activity of cell proliferation.     

Onset of the constrictive effect of indomethacin on the ductus arteriosus in fetal rats.

The time of onset of the constrictive effect of indomethacin on the ductus arteriosus (DA) in fetal rats was assessed by measurement of the caliber of the DA after maternal treatment with indomethacin on days 19-21 of gestation. The day following overnight mating was regarded as day 0 of gestation. Observation was performed by direct exposure of the DA by hand shaving of intact frozen fetuses. On days 20 and 21, the DA was significantly constricted 3 h after maternal treatment with 1 mg/kg of indomethacin. When the DA was examined at 19 1/2 and 19 2/3 days of gestation (3 h after indomethacin exposure), it was significantly constricted at 19 2/3 days but not at 19 1/2 days. Higher doses of indomethacin (10 and 100 mg/kg) induced a significant constriction of the DA at day 19 1/2, but not at the beginning of the same day (1.00 a.m.). These results suggest that the onset of the susceptibility of the DA to the constrictive effect of indomethacin occurs in the first half of day 19 of gestation.     

Active c-erbB-2 induces short-term growth of FDC-P2 cells after IL-3 depletion.

A retroviral expression vector carrying the c-erbB-2 gene was introduced into the FDC-P2 myeloid cell line, which is absolutely dependent on interleukin-3 (IL-3) for proliferation and survival. Since the c-erbB-2 protein appears to be the receptor of an as yet unidentified growth factor, epidermal growth factor receptor (EGFR) was used as a control of a ligand-dependent receptor. FDC-P2 cells expressing normal c-erbB-2 were unable to grow without IL-3 stimulation. The c-erbB-2 protein in these cells was under-phosphorylated on tyrosine residues in vivo. On the contrary, the active c-erbB-2 protein, in which Val-659 was replaced by Glu in the transmembrane domain, and EGF-stimulated EGFR showed significant levels of tyrosine phosphorylation in vivo. These active proteins could promote short-term growth of FDC-P2 cells without IL-3 stimulation, though not indefinitely. These findings suggested that immortalization of this factor-dependent cell line requires an additional oncogenic promoting process(es).