Hideyo Hasumi

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves.

Highly purified ferredoxin-NADP+ reductase from spinach leaves showed at least eight different protein bands in the electrofocused gel. All of them were catalytically active and were adsorbed on a ferredoxin-Sepharose 4B affinity column. The N-terminal amino acid sequence of the main component species was analyzed by the automatic Edman degradation method. It was found that when the reductase was stored at 4 degrees C, new protein bands appeared in isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoreses, but the appearance of the bands was suppressed by the addition of a protease inhibitor, diisopropyl fluorophosphate. This indicates that the molecular heterogeneity of the reductase may result from the digestion with a protease present in spinach leaves.

Kinetic studies on isomerization of ferricytochrome c in alkaline and acid pH ranges by the circular dichroism stopped-flow method

The isomerization of horse-heart ferricytochrome c caused by varying pH was kinetically studied by using circular dichroism (CD) and optical absorption stopped-flow techniques. In the pH range of 7--13, the existence of the three different forms of ferricytochrome c (pH less than 10, pH 10--12, and pH greater than 12) was indicated from the statistical difference CD spectra. On the basis of analyses of the stopped-flow traces in the near-ultraviolet and Soret wavelength regions, the isomerization of ferricytochrome c from neutral form to the above three alkaline forms was interpreted as follows (1) below pH 10, the replacement of the intrinsic ligand of methionine residue by lysine residue occurs; (2) between pH 10 and 12, the uncoupling of the polypeptide chain from close proximity of the heme group occurs first, followed by the interconversion of the intrinsic ligands; and (3) above pH 12, hydroxide form of ferricytochrome c is formed, though the replacement of the intrinsic ligand by extrinsic ligands may occur via different routes from those below pH 12. The CD changes at 288 nm and in the Soret region caused by the pH-jump (down) from pH 6.0 to 1.6 were compared with the appearance of the 620-nm absorption band ascribed to the formation of the high-spin form of ferricytochrome c. Both CD and absorption changes indicated that the isomerization at pH 1.6 consisted of two processes: one proceeded within the dead-time (about 2 ms) of the stopped-flow apparatus and the other proceeded at a determinable rate with the apparatus. On the basis of these results, the isomerization of ferricytochrome c at pH 1.6 was explained as follows: (1) the transition from the low-spin form to the high-spin forms occurs within about 2 ms, the dead-time of the stopped-flow apparatus; and (2) the polypeptide chain is unfolded after the formation of the high-spin form.

Kinetic Analysis of Molecular Interconversion of Immunosuppressant FK506 by High-Performance Liquid Chromatography

High-performance liquid chromatography of FK506, a macrolide immunosuppressant, was performed on a reversed-phase column. The peak was broad with the column kept at room temperature, which was accounted for by slow interconversion between the two forms of FK506. With the use of a heated column, a sharp peak was observed because of the rapid interconversion at high temperature. When the column was cooled to 0°C, two sharp peaks were observed because essentially no interconversion occurred at 0°C during elution. Analysis of the chromatograms obtained at various eluant flow rates indicated that the conversion of the two forms follows first-order kinetics, and the apparent activation energies for the conversions were calculated. The interconvertibility between two molecular forms may be related to the immunosuppressive activity.

Neonatal exposure to endocrine disruptors suppresses juvenile testis weight and steroidogenesis but spermatogenesis is considerably restored during puberty.

Neonatal exposure to endocrine disruptors induces developmental abnormalities in the male reproductive system. As to investigate whether neonatal exposure affects spermatogenesis in juvenile and pubertal testes, Sprague-Dawley rat pups were given various endocrine disruptors by a single injection on the day of birth at concentrations ranging between 4 microM and 40 mM and sacrificed on day 21 (juvenile) or 50 (puberty). The testes were weighed and examined histologically at each stage. Further, the metabolites of steroidogenesis were analyzed using normal-phase high performance liquid chromatography. Neonatal exposure significantly reduced testis weights and steroid biosynthesis of juveniles, but they were highly restored at puberty.

Interconversion of cyclosporin molecular form inducing peak broadening, tailing and splitting during reversed-phase liquid chromatography

SummaryReversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.

Effects of mixed solvents on three elementary steps in the reactions of horseradish peroxidase and lactoperoxidase.

The effects of methanol, acetone, and ethylene glycol (up to 50% v/v) on elementary steps in the reactions of horseradish peroxidase (HRP) and lactoperoxidase (LPO) were studied by means of the stopped-flow method and the difference spectrum. The rate constant (k3,app) of the oxidation reaction of p-cresol with HRP compound II was remarkably reduced in the presence of organic solvents (to 2.3%, 1.8% and 9.4% of the original value in the presence of 50% (v/v) of methanol, acetone and ethylene glycol, respectively), then to a lesser degree were decreased the rate of the oxidation reaction with LPO compound II, and the rate of the oxidation reaction with HRP compound I. These reductions in the reaction rates were not due to competitive inhibition of the solvents, but considered to be related to the degree of exposure of the electron transfer route to the medium. While the rate constant of compound I formation (k1,app) was moderately affected by organic solvents in the case of HRP, the reaction rate with LPO was scarcely affected by organic solvents, being in harmony with the compact heme crevice which probably hampers penetration of solvent molecules. The rate constant (k2,i,app) of the oxidation reaction of an iodide ion by HRP compound I was also hardly affected by the solvents. On the basis of these facts, the mechanism of electron transfer from donors to compound I and compound II is discussed.

Effects of neutral salts on thermal stability of spinach ferredoxin

Summary The effects of various neutral salts on the thermal stability of spinach ferredoxin were studied by means of differential scanning calorimetry and circular dichroism. Upon addition of 1 M NaCl, the apparent denaturation temperature increased by about 13°, and the enthalpy and activation energy for the denaturation also increased by 27% and 44%, respectively. The salt effect was ascribed essentially to the ionic strength of the environment. The circular dichroism measurements showed that the interaction between the iron-sulfur cluster and the protein moiety was modulated by the salt, resulting in stabilization of the protein, It is therefore highly probable that the native ferredoxin structure is maintained through the interaction with the iron-sulfur chromophore.

Studies on the molecular structure of spinach ferredoxin. I. Comparison of two molecular species of ferredoxin

Abstract By hydrophobic chromatography on a Toyopearl HW-60F column, spinach ferredoxin was separated into the major (Fd-I) and minor (Fd-II) components in the native forms. These ferredoxins could be distinguished from each other as two different protein bands on a SDS-polyacrylamide gel electrophoresis in the presence of 4 M urea, but not in the absence of urea. Although both reduced ferredoxins exhibited the EPR spectra of rhombic symmetries having an identical gav value of 1.96 at 22 K, the anisotropy of the g-factors of Fd-II was slightly larger than that of Fd-I; gx = 1.890, gy = 1.955, and gz = 2.043 for Fd-I, and gx = 1.875, gy = 1.962, and gz = 2.049 for Fd-II. In the UV region, the absorption spectrum of Fd-I was subtly different from that of Fd-II. In the visible region, however, the absorption spectrum of Fd-I was essentially identical with that of Fd-II. In contrast, the CD spectra of both ferredoxins were different from each other mainly in the visible region. Upon addition of urea, the respective CD and absorption spectra of both ferredoxins changed in a similar manner. In addition, urea denaturations of both ferredoxins were found to consist of at least three processes from analyses of the time courses of the CD changes. The first and second processes of Fd-I were found to proceed faster than those of Fd-II, while the third process of Fd-I was found to occur slower than that of Fd-II. The iron-sulfur clusters decomposed during the third processes. These results lead to the conclusions that (1) the molecular structure of ferredoxin is maintained through the interaction between the iron-sulfur cluster and the protein moiety, and (2) the structural changes in the protein moiety of ferredoxin result in the geometrical alteration of the cluster.

Purification and properties of multiple molecular forms of yeast peptidyl prolyl cis-trans isomerase

By hydrophobic chromatography on a butyl-Toyopearl 650 M column, yeast peptidyl prolyl cis-trans isomerase (PPIase) was separated into at least three molecular components (PPI-I, PPI-II and PPI-III) in their native forms. On the basis of the result of SDS-PAGE, PPI-II and PPI-III were highly purified and their molecular masses were estimated to be 16.5 and 17.2 kDa, respectively. However, PPI-I was still a mixture of two components with molecular masses of 23.3 and 24.1 kDa. The UV absorption spectrum of PPI-II was slightly different from that of PPI-III. In contrast, the CD spectra of the two proteins were essentially identical in the far-UV region. Upon addition of an immunosuppressant, cyclosporine A (CsA), the absorption spectra of the two highly purified proteins were subtly changed, which was indicative of some alterations in the microenvironments of the aromatic amino-acid residues. The two proteins exhibited subtle but clear differences in the kinetic parameters (kc/Km) for the PPIase-catalyzed cis-trans isomerization and in the inhibition constants of CsA for the PPIase activity. These results lead to the conclusions that (1), a family of PPIases exists in one organism and that (2), one member of the family has multiple molecular forms with different substrate specificities and different affinities for the drugs (inhibitors).

Temperature effects on optical absorption and circular dichroism of cytochrome P-450scc from bovine adrenocortical mitochondria

Temperature-dependent spin changes of the heme iron atom on cytochrome P-450scc were studied by optical absorption and circular dichroism measurements. The optical absorption and circular dichroism spectra of cholesterol-free cytochrome P-450scc did not change between 10 and 26 degrees C. In contrast, the absorbance at 390 nm and the ellipticity at 330 nm of cholesterol-bound cytochrome P-450scc decreased upon temperature elevation, and the absorbance at 424 nm correspondingly increased. These spectral changes were reversible in respect of temperature. The far-ultraviolet circular dichroism spectra of both cholesterol-bound and -free cytochrome P-450scc were not affected by temperature. In addition, bound cholesterol molecule is not released from the cytochrome molecule by increasing temperature. From these results, we propose that temperature modulates specific interactions between the heme protein and bound cholesterol rather than the gross secondary structural changes of the protein.