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Biomedical subjects

T Ando

Publications and source records attributed to T Ando.

At least 649 records · Page 36Linked to original sources
PubMed1982

D-loop cycle. A circular reaction sequence which comprises formation and dissociation of D-loops and inactivation and reactivation of superhelical closed circular DNA promoted by recA protein of Escherichia coli.

Excess recA protein, a protein essential to general genetic recombination in Escherichia coli, promotes a sequence of formation and dissociation of D-loops from negative superhelical closed circular double-stranded DNA (form I DNA) and homologous single-stranded fragments in the presence of excess ATP, resulting in inactivation of the form I DNA without apparent damage to the DNA. The dissociation of D-loops is accompanied by hydrolysis of ATP to ADP that apparently depends on homologous DNA molecules (homology-dependent ATP hydrolysis). However, at a lower concentrations of ATP, we observed anomalous kinetics in the formation and dissociation of D-loops; as the concentration of ATP was decreased, there was a progressively smaller dissociation of D-loops and a faster resynthesis in the second phase, without changing the rate of the first formation of D-loops. This anomaly might suggest that, as the increase in the amount of ADP relative to that of ATP, dissociation form I DNA is stimulated before formation of D-loops is inhibited. We found that addition of ADP inhibited competitively both formation and dissociation of D-loops and that the latter process was more sensitive to the inhibition than was the former process. Addition of a sufficient amount of ADP to inhibit both formation and dissociation of D-loops, cessation of homology-dependent hydrolysis of ATP, or incubation at low temperature resulted in reactivation of form I DNA that had been inactivated by the sequence. In the presence of an ATP-regenerating system, we confirmed our previous result that limiting the amount of recA protein also causes anomalous kinetics in the formation and dissociation of D-loops. These observations indicate that the formation and dissociation of D-loops and the inactivation and reactivation of form I DNA make a circular reaction sequence.

Bacterial Proteins↗
PubMed1982

Isolation and characterization of nuclear basic protein (protamine) from boar spermatozoa.

1. Sperm nuclei were isolated and purified from boar semen by a procedure involving differential solubilization of sperm tail and acellular materials by brief exposure to reducing reagent in the presence of cationic detergent, and sedimentation through 60% sucrose. The weight ratio of DNA:RNA: total protein: protamine in this preparation was 1.00: 0.02: 1.05: 0.75, and the molar ratio of phosphorus to arginine was 1.12. 2. Boar protamine was extracted with cold acid from ethanol precipitate of reduced and carboxymethylated nuclei in 6 M guanidine hydrochloride and purified by ion-exchange chromatography on CM-cellulose. The molecular weight of the protamine was estimated to be 6600 by the gel filtration method. The protamine consisted of a single amino terminus alanine and either half-cystine or arginine as carboxy terminus, and was composed of Thr, Ser3, Pro2, Ala2, Val2, Ile, His, Half-cystine9-10 and Arg26 . 3. Chymotryptic digestion gave rise to a single amino-terminal peptide, Ala-Arg-Tyr, and two carboxy-terminal peptides, Thr-Val-Ile-Arg-Cys-Arg2-Cys and Thr-Val-Ile-Arg-Cys-Arg2, which confirmed the heterogeneity of the protamine at the carboxy-terminal end.

Amino Acid Sequence↗
PubMed1982

Role of superhelicity in homologous pairing of DNA molecules promoted by Escherichia coli recA protein.

In the presence of ATP and an excess of recA protein, superhelical closed circular DNA (form I DNA) and homologous single-stranded fragments paired to form D-loops in the early stage of incubation and dissociated during subsequent incubation. RecA protein that was not bound to single-stranded DNA ("free recA protein") was shown to be responsible for the dissociation of D-loops. Larger amount of free recA protein gave a lower final yield of D-loops. When the concentration of form I DNA was increased in the presence of a fixed amount of single-stranded DNA, larger amounts of free recA protein were required to produce a certain extent of dissociation. When form I DNA, excess recA protein, and ATP were incubated without single-stranded DNA, or with heterologous single-stranded fragments before the addition of homologous single-stranded fragments, formation and subsequent dissociation of D-loops were observed as in the case when all components of the reaction were added from the start. Therefore, the dissociation of D-loops is a result of the stoichiometric interaction between free recA protein and form I DNA bearing D-loops. In the process of formation and dissociation of D-loops, form I DNA was converted to an inactive substrate without any apparent damage to the DNA. The concentration of free recA protein appeared to decrease during the reaction. These observations revealed that formation and dissociation of D-loops are sequential reactions when form I DNA is the substrate and recA protein is present in excess. The dissociation of D-loops and the inactivation of form I DNA can be explained by a model in which recA protein cooperatively binds to form I DNA from the site of D-loop, resulting in stimulation of unidirectional unwinding of the double helix.

Bacterial Proteins↗
PubMed1982

Hydrolysis of ATP dependent on homologous double-stranded DNA and single-stranded fragments promoted by RecA protein of Escherichia coli.

RecA protein is essential to general genetic recombination in Escherichia coli. In the presence of ATP, a stoichiometric amount of recA protein forms D-loops from superhelical closed-circular DNA (form I DNA) and homologous single-stranded fragments, and subsequently dissociates the D-loops. Under appropriate conditions, the hydrolysis of ATP by recA protein depends on the presence of both double-stranded DNA and homologous single-stranded fragments (homology-dependent hydrolysis). In the presence of form I DNA, most of the homology-dependent hydrolysis of ATP by recA protein is related to the dissociation of D-loops rather than the formation of D-loops. RecA protein also promoted the homology-dependent hydrolysis of ATP in the presence of nicked-circular DNA (form II DNA), but unlike the case of form I DNA, this hydrolysis was associated with an increase in the amount of mature D-loops that were detected by the D-loop assay. When double-stranded DNA was superhelical, the homology-dependent hydrolysis of ATP continued at the same rate even after all the D-loops were dissociated. This correlates with our earlier observation that in the process of formation and dissociation of D-loops, form I DNA was converted to an inactive substrate without any apparent damage to the DNA, probably by the formation of a complex with recA protein. All of the observations described above can be explained by a model in which a common mechanism causes dissociation of D-loops from form I DNA, inactivation of form I DNA, and growth of D-loops in form II DNA. The mechanism might involve cooperative binding of recA protein to the duplex DNA from the site of the nascent D-loop, resulting in unidirectional unwinding of the duplex DNA.

Adenosine Triphosphate↗
PubMed1982

Purification and properties of a novel lipase from Staphylococcus aureus 226.

A novel lipase of Staphylococcus aureus 226 was purified by ammonium sulfate precipitation followed by successive chromatographies on hydroxylapatite, Sephadex G-200 and G-150. This method gave 385-fold purification of the enzyme from the culture medium in a yield of 25%. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis and isoelectric focusing. The purified lipase hydrolyzed all the triacylglycerols and 1-monoacylglycerols tested, showing maximal activity with trilaurin and 1-monoolein. Furthermore, 2-monoolein was also found to be one of the best substrates for the lipase. The optimum temperature of the purified enzyme was 60 degrees C with triolein. The molecular weight of the enzyme was reduced in the presence of sodium deoxycholate and was estimated as 34,000 by gel filtration and its isoelectric point as 9.7. Mg2+ and Ca2+ ions enhanced the enzymatic reaction, whereas Mn2+ ions were inhibitory. p-Chloromercuribenzoate, iodoacetamide, and N-ethylmaleimide did not inhibit the enzyme.

Ammonium Sulfate↗
PubMed1982

An improved method for the determination of human blood kinin levels by sensitive kinin radioimmunoassay.

A highly sensitive and specific radioimmunoassay for kinin (minimal detectable amount, 0.5 pg/tube) was applied to measure the blood kinin level. A five ml blood sample was collected with a siliconized needle and plastic syringe which contained 2.5 ml of 0.8 N-HCl. The blood kinin was extracted with butanol, following reextraction with water. According to this procedure, the mean recovery (mean +/- SE) calculated from added 125I-bradykinin (500 CPM) and the known amounts of cold bradykinin were 50.4 +/- 0.8% and 51.1 +/- 2.2%, respectively. In comparison with other sampling methods in 6 normal subjects, the blood samples taken without HCl in syringes showed a higher level (24.4 +/- 10.1 pg/ml) than the samples with HCl (5.3 +/- 1.3 pg/ml). And very high levels were obtained in the plasma samples collected by the method of Talamo or Vinci (0.53 +/- 0.24 ng/ml and 3.5 +/- 1.3 ng/ml, respectively). The kinin content in blood samples taken with HCl was stable at -20 degrees C for at least one month, but increased significantly at room temperature or 4 degrees C for 48 hours. Blood samples were obtained from 17 normal subjects, and 3 patients with acute myocardial infarction. Blood kinin levels in the patient with acute myocardial infarction, 121 +/- 20.9 pg/ml, were significantly higher than those in normal subjects (3.8 +/- 0.5 pg/ml). From these results, it was concluded that high levels of blood kinin reported previously may have resulted from inadequate sampling procedures. Thus, in order to measure blood kinin accurately, inactivation of the kinin generating and destroying enzymes must be done immediately after the sampling. In addition, this radioimmunoassay method should be very useful in investigating the pathophysiological role of blood kinin in various diseases.

Blood Specimen Collection↗
PubMed1981

Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions.

Covalently closed-circular, superhelical SV4O DNA was used in all experiments. EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA. S1 does cleave both strands of superhelical DNA at unbasepaired sites.

DNA Ligases↗