PubMed Health⌕ Search

Biomedical subjects

T Date

Publications and source records attributed to T Date.

At least 73 records · Page 4Linked to original sources
PubMed1990

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding.

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme. In contrast to the wild-type enzyme, which possesses 1 mol of tightly bound NAD+ per mol of enzyme subunit, the mutant enzyme had only 0.05 mol of NAD+ but contained about 0.6 mol each of NADH and adenine per mol of subunit. The mutant enzyme, after removal of the bound compounds by acid-ammonium sulfate treatment, exhibited S-adenosylhomocysteinase activity when assayed in the presence of NAD+. From the appearance of activity as a function of NAD+ concentration, the enzyme was shown to bind NAD+ with a Kd of 23.0 microM at 25 degrees C, a value greater than 280-fold greater than that of the wild-type enzyme. In the presence of a saturating concentration of NAD+, the mutant enzyme showed apparent Km values for substrates similar to those of the wild-type enzyme. Moderate decreases of 8- and 15-fold were observed in Vmax values for the synthetic and hydrolytic directions, respectively. These results indicate the importance of Asp-244 in binding NAD+, and are consistent with the idea that the region of S-adenosylhomocysteinase from residues 213 to 244 is part of the NAD+ binding site. This region has structural features characteristic of the dinucleotide-binding domains of NAD(+)- and FAD-binding proteins (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P.S., Jr., Aksamit, R.R., Unson, C.G., and Cantoni, G.L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 719-723).

Adenosylhomocysteinase↗
PubMed1990

Rat serine dehydratase gene codes for two species of mRNA of which only one is translated into serine dehydratase.

With the previously obtained rat liver serine dehydratase cDNA (SDH2; Ogawa, H., Miller, D.A., Dunn, T., Su, Y., Burcham, J. M., Peraino, C., Fujioka M., Babcock, K., and Pitot, H. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 5809-5813) as a probe, we isolated a different species of cDNA (SDH3) from the same cDNA library from which SDH2 was obtained. Nucleotide sequence analysis has indicated that SDH3 has an open reading frame which encodes 327 amino acid residues and which is identical to that of the cDNA obtained by Noda et al. (Noda, C., Ito, K., Nakamura, T., and Ichihara, A., (1988) FEBS Lett. 234, 331-335). Primer extension analysis and RNase protection mapping clarified that the SDH3 mRNA was the major mRNA for serine dehydratase in the liver, and its transcription begins with a T residue located 23 nucleotides down-stream of a TATA-like box. In vitro transcription/translation experiment demonstrated that SDH3 encoded a polypeptide of 35 kDa, a size in agreement with that of the subunit of the purified protein, whereas SDH2, despite having a size larger than SDH3, produced a peptide of much smaller size that reacted with anti-serine dehydratase IgG. SDH2 was found to have a stop codon early in the sequence and is predicted to encode a polypeptide of 8.9 kDa. Also, SDH2 has a 5'-noncoding sequence different from that of SDH3. These results indicate that alternative transcription initiation and different modes of splicing of the primary transcripts of rat serine dehydratase gene result in the formation of two species of mRNA, of which only one is translated into the mature serine dehydratase protein.

Amino Acid Sequence↗
PubMed1990

There are two major types of hepatitis C virus in Japan.

The polymerase chain reaction (PCR) was used to detect hepatitis C virus (HCV) in plasma from chronic non-A, non-B hepatitis patients. By choice of adequate primers, 19 of 24 samples (79%) were found positive. Sequence analysis of amplified 400 bp cDNA fragments encoding a portion of NS5 gene suggested that HCV can be classified into two types (named K1 and K2) in Japan. Slot blot hybridization of the fragments indicated that 13 were HCV-K1 and 6 were HCV-K2, which show 80% and 67% nucleotide sequence homology, respectively, with that of the prototype.

Amino Acid Sequence↗
PubMed1990

DNA binding activity and transcriptional activator function of the human B-myb protein compared with c-MYB.

Three members of the myb gene family have been identified in human cDNA libraries c-myb, A-myb, and B-myb. We compared the DNA binding properties of the B-myb and c-myb proteins (B-MYB and c-MYB) using bacterially synthesized B-MYB and c-MYB in DNase I footprinting. B-MYB bound to most of the c-MYB binding sites examined, including the c-MYB binding site, MBS-I, in the simian virus (SV) 40 enhancer, in which the most frequent sequence was CCTAACTG. The MBS-I site was an enhancer element dependent on B-MYB and c-MYB in a co-transfection assay that used the B-myb or c-myb expression plasmid. Some sites in the SV40 genome, including the MBS-BI site, had high affinity with B-MYB but little or no affinity with c-MYB, in which the most frequent sequence was AGAAANPyrG. The MBS-BI site was an enhancer element dependent on B-MYB and a very weakly dependent on c-MYB. Our results showed that B-MYB is a transcriptional activator, like c-MYB, and that although B-MYB and c-MYB have similar sequence specificity for DNA binding some sequences were recognized by B-MYB preferentially.

Base Sequence↗
PubMed1990

Construction of Escherichia coli vectors for expression and mutagenesis: synthesis of human c-Myc protein that is initiated at a non-AUG codon in exon 1.

Three types of Escherichia coli vector for both gene expression and mutagenesis were constructed from a plasmid/phage chimera vector pUC118. Each vector contains the lac (pTD-lac), tac (pTD-tac), or T7 promoter (pTD-T7). Downstream from the promoter, these vectors have sequences in common, including a Shine-Dalgarno (SD), multiple cloning sequence, sequence-primer binding site, transcription termination signal, and M13 origin of replication. Using single-stranded circular DNA obtained by infection with helper phage, oligodeoxyribonucleotide (oligo)-directed mutagenesis allows the appropriate fusion between the vector SD sequence and the start codon in the inserted fragment. Since a complementary oligo representing a large deletion is generally used for this construction, the extra nucleotides in the opposing strand form a loop structure. Thus, we have designated this mutagenesis as 'loop-out mutagenesis'. Expression plasmid encoding the larger human c-Myc protein that is initiated at a non-AUG codon in exon 1 and its derivatives were constructed using a pTD-T7 vector. Expression experiments indicated that the wild-type (wt) protein was synthesized poorly after induction with isopropyl-beta-D-thiogalactopyranoside, while one of the derivatives, p62M1T, in which a threonine residue was added at the N terminus of the wt protein, was produced in a large quantity in E. coli.

Base Sequence↗
PubMed1990

Site-directed mutagenesis of recombinant rat DNA polymerase beta: involvement of arginine-183 in primer recognition.

By site-directed mutagenesis using synthetic oligonucleotides, amino acid residues 181Phe-Arg-Arg183 of recombinant rat DNA polymerase beta were replaced by other amino acids to clarify the roles of these residues in the DNA synthesizing reaction. Replacement of Phe-181 by alanine reduced the enzyme activity only 30%. Replacement of Arg-182 by alanine and glutamine resulted in reduction of the activity by about 67% and 95%, respectively. The Arg-182----Gln replacement increased the binding strength to single-stranded DNA but did not significantly change the Km's for the primer and dTTP, suggesting that Arg-182 is involved in modulation of binding to the template rather than to the primer or deoxyribonucleoside triphosphate. Replacement of Arg-183 by Gln resulted in reduction of the activity by about 95%, and this change, although causing little change in binding strength to single-stranded DNA, resulted in a 3-4-fold increase in the Km's for the primer and deoxyribonucleoside triphosphate. A more dramatic change was observed when Arg-183 was replaced by Ala, which resulted in a 99.98% reduction of enzyme activity. Although the Km for deoxyribonucleoside triphosphate of this mutant enzyme was hardly changed, that for the primer increased 159-fold. Therefore, it is concluded that Arg-183 occupies an important part of the primer recognition site of DNA polymerase beta.

Amino Acid Sequence↗
PubMed1990

Application of synthetic DNA probes of human alpha satellite consensus monomer for detection of centromere-involved chromosome abnormalities.

We have synthesized the alphoid monomer of 171 bp based on the consensus sequence of human alpha satellite DNA and constructed a clone of dimeric or tetrameric sequence unit. Southern blot analysis using the clone as a probe showed restriction site periodicities in human DNA digested by EcoRI or BamHI. The synthetic consensus unit could detect the alpha repeated centromeric regions of all human chromosomes by fluorescence in situ hybridization. Using the cells having a dicentric X chromosome, we showed that the two centromeric regions were stained with fluorescent alpha satellite DNA probes. Thus the probe would be useful to detect chromosomal abnormalities such as dicentrics.

Base Sequence↗
PubMed1989

Human liver serine dehydratase. cDNA cloning and sequence homology with hydroxyamino acid dehydratases from other sources.

Rat liver serine dehydratase cDNA was used to screen a human liver cDNA library in lambda gt11. One positive clone occurred in every 5,000 clones. Fifteen positive clones were plaque purified. The largest cDNA obtained contained an open reading frame of 987 base pairs, and 5' and 3' noncoding regions of 89 and 317 base pairs, respectively. The deduced amino acid sequence, with a calculated Mr of 34,615, was similar to that of rat liver serine dehydratase except for the absence of a segment consisting of 36 amino acid residues. In vitro transcription/translation with the cDNA resulted in the formation of a polypeptide with an Mr of approximately 35,000, which cross-reacted with the anti-rat serine dehydratase antibody. These results suggest that the human serine dehydratase is structurally cognate with the rat enzyme. Moreover, portions of the sequence postulated to be essential for activity in microbial threonine dehydratases are found in the mammalian serine dehydratases, suggesting that hydroxyamino and dehydratases may have originated from a common ancestor.

Amino Acid Sequence↗
PubMed1989

Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site.

The cDNA for rat liver S-adenosylhomocysteinase has been cloned, and the nucleic acid sequence has been determined. By comparison of the deduced amino acid sequence for S-adenosylhomocysteinase with that of the dinucleotide binding region for other proteins, the sequence from amino acids 213 to 244 in rat liver S-adenosylhomocysteinase was proposed to be part of the NAD binding site (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). A vector has been constructed that expresses S-adenosylhomocysteinase in Escherichia coli in the presence of isopropyl beta-D-thiogalactopyranoside by inserting the coding sequence of rat liver S-adenosylhomocysteinase cDNA downstream from the lac promoter of plasmid pUC118. The enzyme that is produced comprises as much as 10% of the soluble cellular proteins. The purified enzyme is a tetramer, contains 4 mol of tightly bound NAD, and has kinetic properties indistinguishable from those of the liver enzyme. Tryptic peptide mapping and NH2-terminal sequence analysis indicate that the recombinant enzyme is structurally identical to the liver enzyme except for the absence of the NH2-terminal blocking group. The rat liver enzyme has a blocked NH2-terminal alanine residue (Ogawa, H., Gomi, T., Mueckler, M. M., Fujioka, M., Backlund, P. S., Jr., Aksamit, R. R., Unson, C. G., and Cantoni, G. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 719-723). By oligonucleotide-directed mutagenesis mutant vectors have been generated that express proteins in which each of the glycines in the Gly-Xaa-Gly-Xaa-Xaa-Gly sequence of the putative NAD binding site (residues 219-224) was changed to valine. Immunoblot analysis of extracts of the cells transformed with these vectors reveals the presence of immunoreactive proteins with the subunit molecular weight of S-adenosylhomocysteinase. The mutant proteins have no catalytic activity, contain no bound NAD, and do not form the same quaternary structure as the recombinant S-adenosylhomocysteinase.

Adenosylhomocysteinase↗
PubMed1989

Isolation of human cDNA clones of ski and the ski-related gene, sno.

cDNA clones of ski and the ski-related gene, sno, were obtained by screening human cDNA libraries. The predicted open reading frame of h-ski could encode a protein of 728 amino acid residues. The h-ski protein is highly homologous with the v-ski protein. The overall homology between h-ski and v-ski is 91% at the amino acid level. DNA sequencing analysis revealed two types of cDNA clones from the sno (ski-related novel gene) gene, possibly due to alternative splicing. The first type, named snoN (non Alu-containing), encoded a protein of 684 amino acid residues. The second type, named snoA (Alu-containing), encoded a protein of 415 amino acid residues. The first 366 amino acid residues of snoN and snoA are the same, but subsequent amino acids show divergence. Several transcripts of h-ski (6.0, 4.7, 3.8, 3.0, 2.1 and 1.8 kb) were detected. The mRNAs of h-sno were 6.2, 4.4 and 3.2kb.

Amino Acid Sequence↗
PubMed1989

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex.

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).

Adenosine Triphosphatases↗
PubMed1989

Diurnal change of plasma atrial natriuretic peptide concentrations in patients with congestive heart failure.

Diurnal change of plasma atrial natriuretic peptide (ANP) concentration was observed in 14 patients with congestive heart failure (CHF) and in eight healthy control subjects. Blood pressure, heart rate, and plasma concentration of ANP were obtained at intervals of 4 hours beginning immediately after midnight. In the CHF group, plasma ANP concentrations at the time of blood sampling were all higher than those in the control group. Patients with severe CHF had higher plasma ANP concentrations than those in patients with less severe CHF. Plasma ANP concentration in the control group was highest at 4:00 AM and was lowest at 4:00 PM. The percent change of ANP secretion (% delta ANP): [(ANP at 4:00 AM-ANP at 4:00 PM)/ANP at 4:00 PM] x 100%, increased in the control group, while it decreased in the CHF group. Moreover, % delta ANP was much lower in patients with severe CHF than it was in patients with less severe CHF. There was a possible relation between the severity of CHF and the increase of ANP secretion associated with the relative diminution of nocturnal ANP secretion. Thus the present data imply that the diurnal change in ANP was lost in patients with CHF.

Atrial Natriuretic Factor↗
PubMed1989

Evolution of the vacuolar H+-ATPase: implications for the origin of eukaryotes.

Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70- and 60-kDa subunits of the vacuolar H+-ATPase are approximately equal to 25% identical to the beta and alpha subunits, respectively, of the eubacterial-type F0F1-ATPases. We now report that the same vacuolar H+-ATPase subunits are approximately equal to 50% identical to the alpha and beta subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the F0F1-ATPase beta subunit but is present in the alpha subunit of Sulfolobus. Since the two types of subunits (alpha and beta subunits; 60- and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.

Amino Acid Sequence↗