Haruhiko Noda

Choline uptake systems of rat brain synaptosomes

The uptake of [3H]choline into synaptosomes and the subsequent synthesis of [3H]acetylcholine were examined as functions of choline, Na+ and hemicholinium-3 concentrations in the incubation medium. The results indicate the presence of two carrier-mediated uptake systems for choline. One system requires Na+ for the uptake and the other does not. About 60% of the choline taken up into synaptosomes by the Na+-dependent system were converted to acetylcholine, whereas only a few percent of the choline taken up into synaptosomes by the Na+-independent system were converted to acetylcholine.Km, the choline concentration giving half-maximum transport was 4–8 μM for the Na+-dependent system and about 40 μM for the Na+-independent system.Ki values for hemicholinium-3 were 0.05–0.1 μM for the Na+-dependent system and 40–50 μM for the Na+-independent system.

Isolation and characterization of collagenases I and II from Clostridium histolyticum

Summary 1. Clostridium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.19) was purified by zone electrophoresis on starch and gel-filtration with Sephadex G-200. Two forms of collagenase (I and II) were isolated by subsequent fractionation by DEAE-cellulose column chromatography. 2. These two enzymes hydrolyzed collagen at comparable rates, but synthetic peptides and azocoll at different rates. Collagenase II attacked more bonds of collagen than collagenase I. 3. Sedimentation constant, molecular weight, electrophoretic mobility, and pH optima for the hydrolysis of collagen and synthetic peptide indicated that collagenases I and II are different enzymes. However, their amino acid compositions were similar.

Reversible thermal unfolding of thermostable cytochrome c-552

Abstract Reversible thermal denaturation of cytochrome c -552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation ( ΔG ) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.

Reactivation kinetics of guanidine denatured bovine carbonic anhydrase B

Abstract Denaturation and reactivation of bovine carbonic anhydrase B was studied with particular attention to the anomalous behavior in the transition region (about 2 m guanidine hydrochloride) that had been reported by previous workers. The denaturation curve based on the partition coefficient of gel chromatography was markedly different from the one based on the ultraviolet difference spectroscopy. Intrinsic viscosity and fluorescence intensity were also measured in guanidine solution. Reactivation of esterase activity of the enzyme was over 90% complete with an average half time of 9 ± 1 min when the protein was fully denatured in 5 m guanidine hydrochloride. Similar reactivation from 2 m guanidine solution showed the dependence of the extent of final activity regain on the time of incubation in 2 m guanidine solution. It decreased to a plateau value of 40–50% of the native activity after 24 h in 2 m guanidine. The irreversibly inactivated fraction could be reactivated if it was transferred to 5 m guanidine before reactivation experiment. Renaturation kinetics followed by ultraviolet difference spectroscopy showed a fast phase ( t 1 2 sec ) and a slow phase ( t 1 2 = 7 ± 1 min ).

Aggregation of myosin A

Abstract Myosin A molecules aggregate and form polymers of about 1 μ in length, when the ionic strength of pH of their solution is lowered. In 0.2 M KCl, myosin A is fulully dissociated at pH 7.3 and higher, but fully polymerized at pH 6.5 and lower. When myosin A solution is in an intermediate state of aggregation, addition of ATP causes dissociation. The birefringence of myosin A solution corrected to full orientation and unit concentration is 1.2·10 −2 .

Natural F-actin: I. Direct isolation of F-actin from myofibrils and its physico-chemical properties

Abstract 1. 1. F-actin was directly isolated from myofibrils without the use of any depolymerization process or acetone treatment. 2. 2. Natural F-actin was very similar to Straub F-actin with respect to its interaction with myosin. 3. 3. The particle length calculated for natural F-actin was shorter than that of Straub F-actin, and corresponded with the possible unit length of the I-filaments in a sarcomere (approx. 1–2 μ). 4. 4. Natural F-actin differed from Straub F-actin in its poor intermolecular interaction or lack of network formation. It had an intrinsic viscosity of about 4 (100 ml/g) and a sedimentation coefficient, s 20, w 0 , of about 40 S.

Studies on silk fibroin of Bombyx mori directly extracted from the silk gland II. Effect of reduction of disulfide bonds and subunit structure

Abstract The fibroin directly extracted from silk glands of Bombyx mori was redcued with 2-mercaptoethanol in a 6 M guanidine·HCl or 8 M urea solution of pH 7.0. The effect of reduction was followed by methods such as gel electrophoresis with sodium dodecyl sulfate, gel filtration by Sephadex G-200, sedimentation equilibrium, and viscosity measurement. It was indicated that the fibroin molecule was composed of three small components of molecular weight 2.6·104 and one large component of molecular weight 2.8·105, connected by disulfide bonds.

Physico-chemical studies on the soluble collagen of rat-tail tendon

Abstract From an extraction solution of rat-tail tendon with dilute acetic acid, a major protein component which was assumed to be responsible for reprecipitating striated fibers was purified. By measuring sedimentation constant, viscosity and diffusion constant, its molecular weight and shape were calculated. The molecular weight was found to be about 700, 000. Assuming the molecules to be prolate ellipsoids of revolution, the long axis was calculated to be 5, 000 A and the short axis 18 A.

Study on the photochemical reaction of HCN and its polymer products relating to primary chemical evolution

The photochemical reaction of HCN at 184.9 nm is studied in the gas phase. (CN)2, H2, CH4, NH3, N2H4, C2H6, and CH3NH2 are identified as gas phase products, and a reaction mechanism is proposed. HCN polymers** are also obtained as solid reaction products, and their structure is investigated by Infrared Spectroscopy, UV-Visible Spectroscopy, Mass Spectrometry, and Amino acid Analysis. The process and nature of the formation of the polymers are discussed.

Studies on silk fibroin of Bombyx mori directly extracted from the silk gland: I. Molecular weight determination in guanidine hydrochloride or urea solutions

Abstract The weight average molecular weight of fibroin directly extracted from the posterior portion of the middle silk gland of Bombyx mori was determined in 6 M guanidine · HCl or 8 M urea solution at pH 7.0. A value of 3.65·10 5 ± 0.20·10 5 was obtained. The fibroin molecule is homogeneous with respect to its molecular weight. The fibroin molecule is not composed of subunits bound by noncovalent bonds, because the molecular weight measured in a dilute salt solution and that in 6 M guanidine·HCl or 8 M urea did not differ. The weight average molecular weight of fibroin decreased in an alkaline solution.