Deep water pathways in the North Pacific Ocean revealed by Lagrangian particle tracking.

Lagrangian particle tracking experiments are conducted to investigate the pathways of deep water in the North Pacific Ocean. The flow field is taken from a state-of-the-art deep circulation simulation. An unprecedented number of particles are tracked to quantify the volume transport and residence time. Half of the North Pacific deep water returns to the Southern Ocean, and its principal pathway is along the western boundary current in the Southwest Pacific Basin in the deep layer. About 30% is exported to the Indian Ocean after upwelling to the shallow layer in the western North Pacific Ocean. The rest is transported to the Arctic Ocean through the Bering Strait or evaporates within the Pacific Ocean. Upwelling of deep water is confined in the western North Pacific Ocean owing to the strong vertical mixing. The mean residence time of deep water in the North Pacific Ocean is estimated to be several hundred years, which is consistent with the observed radiocarbon distribution.

K201 (JTV-519) alters the spatiotemporal properties of diastolic Ca(2+) release and the associated diastolic contraction during ß-adrenergic stimulation in rat ventricular cardiomyocytes.

K201 has previously been shown to reduce diastolic contractions in vivo during ß-adrenergic stimulation and elevated extracellular calcium concentration ([Ca(2+)](o)). The present study characterised the effect of K201 on electrically stimulated and spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca(2+) release and contractile events in isolated rat cardiomyocytes during ß-adrenergic stimulation and elevated [Ca(2+)](o). Parallel experiments using confocal microscopy examined spontaneous diastolic Ca(2+) release events at an enhanced spatiotemporal resolution. 1.0 µmol/L K201 in the presence of 150 nmol/L isoproterenol (ISO) and 4.75 mmol/L [Ca(2+)](o) significantly decreased the amplitude of diastolic contractions to ~16% of control levels. The stimulated free Ca(2+) transient amplitude was significantly reduced, but stimulated cell shortening was not significantly altered. When intracellular buffering was taken into account, K201 led to an increase in action potential-induced SR Ca(2+) release. Myofilament sensitivity to Ca(2+) was not changed by K201. Confocal microscopy revealed diastolic events composed of multiple Ca(2+) waves (2-3) originating at various points along the cardiomyocyte length during each diastolic period. 1.0 µmol/L K201 significantly reduced the (a) frequency of diastolic events and (b) initiation points/diastolic interval in the remaining diastolic events to 61% and 71% of control levels respectively. 1.0 µmol/L K201 can reduce the probability of spontaneous diastolic Ca(2+) release and their associated contractions which may limit the propensity for the contractile dysfunction observed in vivo.

Conformational change in a single molecular species, beta3, of beta-conglycinin in acidic ethanol solution.

Several physicochemical experiments were done to obtain further information on the conformational changes occurring in beta-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, beta3. By far-UV circular dichroism (CD), a transition from beta-sheet to alpha-helical structure was observed upon addition of acidic-ethanol, and the alpha-helix content was found to reach 76% in 70% ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the beta3 species was significantly altered at ethanol concentrations between 10 and 20%. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the beta3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the beta3 molecules were found to aggregate with an increase in ethanol concentration.

A simplified procedure for purification of cytochrome P-450 by preparative ampholine gel for isoelectric focusing.

The purification process for cytochrome P450 is very complicated, involving five or more column chromatography steps for the final preparation. This paper describes a reduction in the number of the steps; it can be easily purified from pig testis microsomes with improved the yield. As the first step, DEAE-Toyopearl column chromatography is performed only once and then, as the second step, the partially purified cytochrome P450 is completely purified by a preparative Ampholine PAG-plate Gel for Isoelectric Focusing. The combination reduced the purification to a two-step procedure.

Two-step purification of cytochrome P-450 from adult pig testis by isoelectric focusing.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing preparative isoelectrofocusing. Purification was achieved 1132 times with a yield of 4.82%. 17alpha-hydroxylase activity was shown to be 14.5 nmol of product/min/nmol of P-450. The cytochrome P-450 was determined to have an isoelectric point of 6.45 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450.

Purification of cytochrome P-450 from adult pig testis by hydroxylapatite and deoxycorticosterone affinity column chromatography.

Adult testicular cytochrome P-450 was purified by a two-step procedure utilizing hydroxylapatite and deoxycorticosterone affinity column chromatography. Cytochrome P-450 was determined to have an isoelectric point of 6.5 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular mass was estimated to be 52000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited the absorption spectrum of a typical cytochrome P-450. A 1000-fold purification was achieved with a yield of 5%.

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing.

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.

Purification of cytochrome b5 from pig testis microsomes by isoelectric focusing in an immobiline pH gradient.

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.

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.

Effect of temperature on molecular structure of cyclosporin A.

By reversed-phase HPLC and NMR spectroscopy, cyclosporin A (CsA) was found to have several molecular structures in polar solvents. In addition, temperature change was found to cause the structural alteration in the polypeptide chain of CsA. Although CD spectrum of CsA in ethanol/water was indicative of the beta-turn structure rather than the beta-sheet structure of the polypeptide chain, the temperature-dependent structural changes were undetectable by CD spectroscopy. The inhibitory effect of CsA on peptidyl prolyl cis-trans isomerase activity was suppressed with elevation in temperature. These results indicate that the alteration in the tertiary structure of CsA plays an important role for the binding capability to its receptor and the immunosuppressive activity.

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 degree C, two sharp peaks were observed because essentially no interconversion occurred at 0 degree 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 the two molecular forms may be related to the immunosuppressive activity.

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).

Catalysis of proline isomerization during protein-folding reactions.

The enzyme peptidylprolyl cis-trans isomerase (PPI) is known to catalyze proline isomerization in short proline-containing peptides. If PPI can be shown to generally catalyze isomerization of proline residues in proteins, then it would be a valuable diagnostic reagent for recognition of isomerization, which has proven to be extremely difficult to characterize by other methods. In this study, the catalytic effect of PPI on the slow refolding reactions of seven different proteins has been studied, and in only two cases (RNase T1 and cytochrome c) could significant catalysis be seen. PPI also caused no enhancement in the rate for the 'subtle' conformational changes of native concanavalin A or native Fragment I of prothrombin, which have been suggested to be rate-limited by proline isomerization. There was a small effect of PPI observed for the generation of native RNAase A from the fully-reduced form when the glutathione concentration was low. The conclusion from these studies is that PPI can weakly catalyze some protein processes which are rate-limited by proline isomerization, but probably exhibits no measureable catalysis toward others. This somewhat limits the usefulness of PPI as a diagnostic reagent for proline isomerization.

Spectrophotometric studies on alkaline isomerization of spinach ferredoxin.

The gross protein structure, the microenvironment of the iron-sulfur cluster, and the effect of neutral salts on the molecular structure of spinach ferredoxin were studied by CD and absorption spectroscopy in the alkaline pH range. In the pH range of 7-11, the existence of reversible isomerization which consisted of at least two proton dissociation processes was indicated by the statical CD and absorption spectra. The CD changes in the visible and far-UV regions were dramatic upon elevation of the pH from neutral to alkaline, indicating a significant alteration of the microenvironment of the cluster and a decrease in the ordered secondary structures. The absorption change in the visible region due to pH elevation was small but clearly observed with a high signal-to-noise ratio. The numbers of protons involved in the respective processes and the apparent pK values obtained from the pH-dependence of the CD changes were in good agreement with those obtained from the pH-dependence of the absorption changes in the visible region. In addition, the rate constants obtained from the time courses of the CD and absorption changes agreed with one another. By the addition of 1 M NaCl, the CD and absorption spectra at alkaline pH were reversed almost to those at neutral pH without significant pH change. On the other hand, above pH 11, ferredoxin was found to be irreversibly denatured. Based on analyses of the statical CD and absorption spectra and of the time courses of the CD changes, the probable mechanism of the isomerization was considered to be as follows: (Formula: see text) where H stands for a proton, N-form for native ferredoxin at neutral pH, N*-form for alkaline ferredoxin below pH 11 which still has the iron-sulfur cluster but with disordered secondary structures of the polypeptide chain, and D-form for completely denatured ferredoxin above pH 11. These results lead to the conclusions that (1) the interaction between the protein moiety and the iron-sulfur cluster is essential for maintaining the native ferredoxin structure, and (2) neutral salts protect the polypeptide chain from unfolding through electrostatic interaction with the ionized side chains, resulting in the stabilization of ferredoxin.

Heme-linked spectral changes of the protein moiety of hemoproteins in the near ultraviolet region.

Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.

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.

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.

Analyses of optical absorption and circular dichroism spectra of spinach ferredoxin at alkaline pH.

The whole protein structure and the microenvironments of the iron-sulfur cluster and of the side chains of amino acid residues of spinach ferredoxin were studied by optical absorption and circular dichroism (CD) spectroscopy in the alkaline pH range. From the pH-dependence of the optical absorption changes at 245 nm, the four tyrosyl residues of ferredoxin were classified into three groups: one exposed residue with a normal apparent pK value of 10.1, two exposed residues with abnormal apparent pK values of 12.0, and one buried residue showing time-dependent ionization. The absorption in the visible region disappeared gradually with the ionization of the buried residue rather than that of the three exposed residues. The apparent pK value of 10.0 was obtained from the rapid CD changes at 258 nm caused by pH elevation from neutral to alkaline pH. The structural alteration associated with the CD change had no effect on the secondary structure of the protein moiety other than the iron-sulfur cluster and the microenvironment of the cluster. The rate constants obtained from the time courses of the CD changes in the near-ultraviolet and visible regions were in good agreement with those obtained from the time courses of the optical absorption changes. These results lead to the conclusions that (1) the native ferredoxin structure is maintained through the interaction with the iron-sulfur cluster and (2) the protein structure in the neighborhood of the cluster, important for the physiological activity, is not perturbed even though the exposed tyrosyl residues are ionized.

Kinetic studies on the reconstitution of deoxyhemoglobin from isolated alpha and beta chains.

The reconstitution reaction of deoxy hemoglobin (Hb) tetramer from isolated alpha and beta chains was kinetically studied by measuring circular dichroism (CD) changes in the Soret and the ultraviolet regions and optical absorbance change in the Soret region with a stopped-flow apparatus. The CD change in the Soret region was a fast reaction, followed by that in the ultraviolet region and the absorbance change in the Soret region. This fast reaction followed a simple second-order rate law with a rate constant of 6.4 x 10(5) M-1 . s-1. These results indicated that the combination of alpha and beta monomers into an alpha beta dimer accompanied the CD change in the Soret region and was the rate-limiting step of the overall reconstitution reaction of deoxyHb tetramer. On the other hand, the CD change in the ultraviolet region was ascribed to the combination of two alpha beta dimers into an Hb tetramer. The absorbance change in the Soret region was related to both the combination of alpha and beta monomers and that of two alpha beta dimers. From the analyses of these reactions the rate constant of the combination of two alpha beta dimers was determined to be 1.0 x 10(6) M-1 . s-1.

Further physicochemical studies on the complex formation between iron-sulfur proteins and flavoproteins from spinach chloroplast and beef adrenal cortex electron-transfer systems.

Physicochemical experiments were performed in order to get further evidence of the formation of complexes between iron-sulfur proteins and flavoproteins from spinach chloroplast and beef adrenal cortex electron-transfer systems. With both spinach and adrenal iron-sulfur protein-flavoprotein systems, characteristic difference circular dichroism (CD) spectra between the sum of CD spectra of the respective proteins and the CD spectrum of a mixture with a molar ratio of one to one were obtained in the visible and far-ultraviolet regions. By differential scanning calorimetry (DSC) measurements, ferredoxin and ferredoxin-NADP reductase were found to be stabilized against heat treatment upon mixing: (1) the apparent denaturation temperatures of ferredoxin and the reductase increased by about 18 and 5 degree C, respectively, and (2) the activation energy of the denaturation reaction increased by about 50%. These results lead to the conclusion that the iron-sulfur protein and flavoprotein from a tightly bound complex, and suggest that the complex formation alters both the environments of the iron-sulfur and flavin chromophores and the contents of the ordered structures, resulting in stabilization of the proteins.