An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Heavy chain of human high molecular weight and low molecular weight kininogens binds calcium ion

An antibody subpopulation, anti high molecular weight (anti-HMW) kininogen-Ca2+ antibody able to bind specifically to the HMW kininogen-Ca2+ complex, was isolated from anti-HMW kininogen antiserum. Partially purified anti-HMW kininogen antibody was applied to a HMW kininogen-Sepharose column equilibrated with 40 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 7.5, containing 1.0 M NaCl and 1 mM CaCl2, and anti-HMW kininogen-Ca2+ antibody was eluted with 5 mM ethylenediaminetetraacetic acid. As a result of characterization by enzyme-linked immunosorbent assay, this antibody specifically recognized the cyanogen bromide cleaved fragment 1 (CB-1) region (1-160 amino acid sequence) of the heavy chain of kininogen molecules in the presence of Ca2+ or Mg2+. Furthermore, circular dichroism (CD) experiments showed that the conformational changes of HMW kininogen and heavy chain were induced by metal ions such as Ca2+ and Mg2+ and that these changes were due to the conformational change of the CB-1 region of the heavy chain. The dissociation constant (Kd) for the heavy chain-Ca2+ measured by CD analysis at 214 nm was found to be 0.33 +/- 0.09 mM (mean +/- SD). The number of Ca2+-binding sites of heavy chain calculated from the Hill plot was 1.15 +/- 0.04 (mean +/- SD). Then, a possible Ca2+-binding site was found in the amino-terminal portion of the heavy chain of kininogen molecules.     

Nucleotide sequence of Suncus murinus immunoglobulin mu gene and comparison with mouse and human mu genes

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [-³³P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly⁸⁴³ and Met⁸⁹⁵ of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerases. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Cloning of cDNA encoding human H-protein, a constituent of the glycine cleavage system

A cDNA that encodes human H-protein, a constituent protein of the glycine cleavage system, was cloned with anti-rat H-protein antibody as a probe from a human liver cDNA library constructed with an expression vector, lambda gt11. The longest size of cDNA of the isolated clones was about 750 base long (lambda HH15B9). On the other hand, we determined the primary structure of human H-protein from the amino terminal Ser by the 12th Val, including a hexapeptide, -Glu-Lys-His-Glu-Trp-Val-. In addition to the finding that most cDNA inserts cloned hybridized with the synthetic DNA probe composed of the possible sequences for the hexapeptide, we confirmed that lambda HH15B9 encodes the partial primary structure of H-protein in an open reading frame.     

Conservation of DNA sequences for plasmid-mediated citrate utilization within the enterobacteria.

Southern blot DNA-DNA hybridization experiments with a cloned Cit+ DNA fragment as a probe showed that the plasmid-mediated Cit+ determinants from four Cit plasmids (R726, pOH3001, pOH3035, and pOH30221) were all homologous. Sequences homologous to the plasmid-borne Cit+ gene were also found in total bacterial DNA isolated from Salmonella paratyphi B, Salmonella enteritidis, Salmonella typhimurium LT-2, Citrobacter freundii, ATCC 8090, Citrobacter amalonaticus ATCC 25405, Klebsiella pneumoniae I and IID 977, and Enterobacter aerogenes ATCC 13048. The DNA digest from C. amalonaticus ATCC 25405 contained a 1.4-kilobase BamHI-HincII DNA fragment that was strongly homologous with and identical in size to the plasmid Cit+ probe.     

Nucleotide sequence of the gene determining plasmid-mediated citrate utilization.

The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Spontaneous deletion of citrate-utilizing ability promoted by insertion sequences.

The citrate utilization (Cit+) transposon Tn3411 was shown to be flanked by directly repeated sequences (IS3411L and IS3411R) by restriction enzyme analysis and electron microscope observation. Cit- deletion mutants were frequently found to be generated in pBR322::Tn3411 by intramolecular recombination between the two copies of IS3411. The flanking IS3411 elements of Tn3411 were shown to be functional insertion sequences by Tn3411-mediated direct and inverse transposition. Tn3411-mediated inverse transposition from pBR322::Tn3411 to the F-plasmid derivative pED100 occurred more efficiently than that of direct transposition of the Cit+ determinant. This was thought to be due to the differential transposability of IS3411L and IS3411R in the transposition process. The frequency of transposition of IS3411 marked with a chloramphenicol resistance determinant was much higher than IS3411-mediated cointegrate formation, suggesting that replicon fusions are not essential intermediates in the transposition process of Tn3411 or IS3411. Spontaneous deletions occurred with high frequency in recA hosts. The spontaneous deletion promoted by homologous recombination between two IS3411 elements in Tn3411 was examined with deletion mutants.