Protein-coated beta-ferric hydrous oxide particles. An electrokinetic and electrooptic study.

Using microelectrophoresis and electric light scattering techniques, we investigated the adsorption characteristics, surface coverage and surface electric parameters of superstructures from two isoforms of plastocyanin, PCa and PCb, in an oxidized state adsorbed on beta-ferric hydrous oxide particles. The surface electric charge and electric dipole moments of the composite particles and the thickness of the protein adsorption layer are determined in a wide pH range, at different ionic strengths and concentration ratios of PC to beta-FeOOH. The adsorption of the two proteins was found to shift the particles' isoelectric point and to alter the total electric charge and the electric dipole moments of the oxide particles to different extent. A "reversal" in the direction of the permanent dipole moment is observed at lower pH for PCb- than for PCa-coated oxide particles. Strict correlation is found between the changes in the electrokinetic charge of the composite particles and the variation in their "permanent" dipole moments. Data suggest that the adsorption of the proteins is driven by electrostatic and/or hydrophobic interactions with the oxide surfaces dependent on pH. The adsorption behaviour is consistent with the involvement of the "eastern" and "northern" patches of the plastocyanin molecules in their adsorption on the oxide surfaces that are differently charged depending on pH.

Extra- and intra-cellular lytic effects of Cytophaga sp. LR2 on the red microalgae Rhodella reticulata.

AIMS: To evaluate the lytic activities of crude enzymes from Cytophaga sp. LR2 on Rhodella reticulata cells and isolated algal polysaccharide. METHODS AND RESULTS: The Cytophaga compartment was separated after centrifugation in a cell suspension for 30 min at 18,000 g. The extracellular enzyme was obtained from the supernatant and the intracellular from the pelleted cells after sonication and removal of debris. Algal cells were incubated with extra- or intracellular preparations and sowed onto agar medium. The suppressive effect of the extracellular enzyme on colony-forming units was found to be almost twice as high. The result was still more pronounced when treated cells had been shocked osmotically before seeding. Saccharolytic activity was evaluated by changes in the reducing sugars in the media. Concerning isolated algal polysaccharide, the reducing power of the two bacterial preparates was relatively low. A combined fraction showed the highest lytic activity. Using native and SDS electrophoresis some relation between the prevalence of the extra and intracellular protein patterns was registered. Two of the common components' molecular weight masses of 50 and 21 kDa were found to be reproducible in native- and SDS-containing gel. CONCLUSIONS: Cytophaga sp. LR2 produce extra- and intracellular enzymes active in destroying Rhodella cultures. The agents excreted in the medium are more effective.We suppose that two or three different classes of enzymes are involved in the lysis process. The comparative electrophoresis in this case shows the protein components with predictable functions. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining different simple and reproducible approaches to identify the lytic capability of Cytophaga sp. LR2 on R. reticulata.

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability.

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.

Twin plastocyanin dimorphism in tobacco.

Two iso-plastocyanin fractions, oxidized b-plastocyanin, PCb(II) and reduced a-plastocyanin, PCa(I), have been isolated from whole tobacco leaves by conventional chromatography on DEAE-cellulose. The isoelectric points of PCa and PCb at 10 degrees C were found to be 3.99 and 3.97, respectively. When the primary structures were analysed, a microheterogeneity within both PCa and PCb was observed. By appropriate peptide arrangements the amino-acid sequences of two PCa (PCa' and PCa") and two PCb (PCb' and PCb") have been differentiated. All four sequences contain 99 amino-acid residues. PCa' and PCa" differ in one position, where Ser-58 in PCa' is replaced by Pro in PCa".PCb' and PCb" differ in three positions, where Gly-65, Thr-81 and Ala-85 in PCb' are replaced by Ala, Ser and Ser in PCb", respectively. PCa (PCa'/PCa") generally differs from PCb (PCb'/PCb") in three positions, where Val-52, Glu-61 and Tyr-62 in PCa'/PCa" are replaced by Ala, Asp and Leu in PCb'/PCb", respectively. Fluorescence spectra of oxidized tobacco PCa and PCb have been characterized with an emission-maximum position at around 340 nm. The presence of one extra tyrosyl (Tyr-62) in PCa results in a weak increase of the maximal intensity in conjunction with a slight blue-shift of the maximum position.

Microheterogeneity of parsley plastocyanin.

A procedure for isolation of two iso-plastocyanins from parsley has been described here. Three consecutive chromatographic steps on DE-52-Whatman cellulose were applied for isolation of two total plastocyanin (PC) fractions, oxidized [PC(II)] and reduced [PC(I)]. By chromatofocusing of PC(II) on Polybuffer exchanger 74 two different plastocyanins, designated as plastocyanin a (PCa) and plastocyanin b (PCb), were obtained. The isoelectric points (pI) of PCa and PCb at 10 degrees C are 4.16 and 4.14, respectively. The complete amino acid sequences of PCa and PCb were determined. The two iso-proteins consist of 97 amino acid residues and differ only at sequence position 53, where Glu in PCa is replaced by Asp in PCb.

[Triiodothyronine binding by the liver nuclei and mitochondria]. / Sviazyvanie triiodtironina iadrami i mitokhondriiami pecheni.

The binding of I125-triiodothyronine by male thyroidectomized rat liver nuclei and mitochondria in vivo and in vitro was studied. Labeled triiodothyronine was bound by the liver nuclei and mitochondria proteins. 90% radioactivity was bound by the nuclear nonhistone proteins. The binding of I125-triiodothyronine to the nuclei and mitochondria protein receptors was inhibited by unlabeled triiodothyronine and ICl. It is suggested that aromatic amino acids serve as the binding sites of the protein receptors, and that iodine atoms in the thyroid hormone molecules participated directly in the binding process.

[Effect of thyroid hormones on protein synthesis in microsomes in vitro]. / Deistvie tireoidnykh gormonov na sintez belka v mikrosomakh in vitro.

I2 and ICl were shown to possess the thyroxin-like effect on protein synthesis in microsomes, isolated from liver tissue of young thyroidectomized rats and incubated in the medium containing 3H-glycine and 1-14C-leucine. Triiodothyronine and iodine-containing substances increased 3.5--3.9-fold the incorporation of these labelled amino acids into microsomal proteins as compared with untreated microsomes from thyroidectomized rats. Olivomycin and cycloheximide abolished the stimulating effect of T3 and iodine ions on the protein synthesis in microsomes. ICl exhibited a distinctly shorter, as compared with T3, latent period of action on the protein synthesis in microsomes of thyroidectomized animals.

[Effect of triiodothyronine and ICl on protein synthesis in cell-free systems]. / Vliianie triiodtironina i ICl na sintez belka v beskletochnykh sistemakh.

Iodine ions exhibited the thyroxin-like effect on incorporation of 1-14C-leucine into proteins of isolated mitochondria and microsomes of thyroidectomized rats in vitro. Thyroxin, triiodothyronine (T3) and ICl increased the incorporation of 1-14C-leucine into proteins of isolated mitochondria of thyroidectomized rats, but did not affect the protein synthesis in microsomes in vitro. Rifampycin and olivomycin abolished completely the stimulating effect of T3 and ICl on incorporation of the label into mitochondrial proteins. The thyroid hormones and iodine ions stimulated protein synthesis in vitro in liver microsomes of thyroidectomized animals only after preincubation with mitochondria or nuclei. In these conditions preincubation with mitochondria elevated the rate of 1-14C-leucine incorporation into microsomal proteins 2--2.5-fold. In similar experiments with nuclei--4--4.8-fold stimulation was detected. Thyroid hormones and iodine ions stimulated synthesis of specific factors in mitochondria (MBS) and in nuclei (NBS) of thyroidectomized rat liver tissue, which increased the protein synthesis in isolated microsomes in vitro. Synthesis of MBS- and NBS-factor required the presence of all the four ribosetriphosphates (ATP, GTP, UTP, CTP) and was inhibited completely by olivomycin; rifampycin blocked only the MBS factor synthesis. NBS- and MBS-factors appear to be RNA (mRNA), synthesized in nuclei and mitochondria, which are transported into the incubation media and translated by ribosomes.

[Effect of in vivo thyroid hormones and IGL on protein synthesis in the mitochondria of thyroidectomized animals]. / Vliianie in vivo tireoidnykh gormonov i IGL na sintez belka v mitokhondriiakh tireoidéktomirovannykh zhivotnykh.

The authors studied the effect of triiodothyronine (T3) and IGL on the intensity of L-14C-tyrosine incorporation, and on the rate of protein of synthesis in the liver mitochondria of thyroidectomized rats, as well as on radioactivity of the liver amino acid poll. It was found that the intensity of L-14C-tyrosins incorporation into the protein of the liver mitochondria in thyroidectomized animals and the rate of protein synthesis in them was half that in sham-operated animals. T3 or IGL administration to thyroidectomized rats normalized protein synthesis in the liver mitochondria. According to all the biochemical indices studied the IGL effect was analogous to that of triiodothyronine. The absence of thyroid hormones in the organism of thyroidectomized animals, or T3 or IGL administration had no effect on the radioactivity of the free tyrosine pool in the liver tissue.

[Effect of thyroid hormones and iodine ions on the amino acid incorporation into proteins of liver mitochondria]. / Vliianie tireoidnykh gormonov i ionov ioda na vkliuchenie aminokislot i belki mitokhondrii pecheni.

The thyroxin-like effect of I2 and ICl on the 3H-glycine and and 14C-leucine incorporation into proteins of mitochondria isolated from the liver of thyroidectomized animals was demonstrated. Triiodthyronine and iodine-containing substances increased in vitro incorporation of labeled amino acids into proteins of isolated mitochondria. The stimulating effect of T3 and ICl was eliminated by olivomycine and chloramphenicol. The action of ICl in these reactions had a much shorter latent period in comparison with the T3 action. The effect of IC1 was expressed as soon as the 30th minute after the injection. The results obtained confirmed the supposition that under definite conditions iodine ions could imitate the effect of the thyroid hormones on the protein synthesis in the cell of animals; the problem of a possibility of thyroid hormones to realize its biological effect at the moelcular level with the aid of iodine ions is thus put forward.