Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

Quasi-elastic light scattering of Artemia ribosomes sedimented in a CsCl density gradient

It is impossible to measure the diffusion coefficient of macromolecules directly and accurately by quasi—elastic light scattering, when aggregates cannot be eliminated from the solutions to be investigated. Nevertheless, a simple method can be applied to overcome this problem in many cases. Aggregates are separated from the monomeric macromolecules by rate-zonal sedimentation in a CsCl density gradient in a transparent centrifugation tube; the monomers are then located by laser light scattering intensity measurements; photon correlation spectroscopy of the scattered light finally yields their diffusion coefficient. The viscosity of aqueous CsCl solutions at different temperatures and concentrations allows a good separation by centrifugation and a low uncertainty in the reduction of the measured diffusion coefficient to standard conditions.The application of the method to eukaryotic large ribosomal subunits is described as an example.     

Opiate binding properties of naturally occurring N- and C-terminus modified beta-endorphins

Beta-endorphin is further processed within the pituitary and brain by either N-terminal acetylation, carboxy-terminal proteolysis, or both. These naturally occurring analogues are stored intracellularly and, in some tissues, represent the majority of beta-endorphin immunoreactivity detected by antisera. It is therefore critical to determine their relative potencies at the opiate receptor. This study demonstrates that cleavage of the C-terminus tetrapeptide brings about a 10-fold decrease in opiate binding potency of either camel or human beta-endorphin. N-Acetylation, on the other hand, causes over a thousand fold loss in opiate potency rendering the peptide effectively inactive. Since unmodified beta-endorphin is approximately equipotent at multiple opiate receptors, we tested for possible differential shifts towards mu or delta-type receptors which may result from the modification. Our results show no change in selectivity, but simply an overall loss of potency.     

Two new C-methylated flavonoids from Myrica gale

From the leaves of Myrica gale 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone has been isolated. The fruits yielded 2′-hydroxy-4′,6′-dimethoxy-3′-methyldihydrochalcone. The constitutions were deduced from spectroscopic data and confirmed by synthesis.     

The systematic implications of the complexity of leaf flavonoids in the bromeliaceae

In a leaf survey of 61 species of the Bromeliaceae, an unexpectedly wide spectrum of flavonoid constituents was encountered. The family is unique amongst the monocotyledons in the frequency and variety of flavonoids with extra hydroxylation or methoxylation at the 6-position. More common flavonols (in 43% of species) and flavones (in 13%) are distributed throughout the family whereas the rarer flavonoid classes are restricted to one or two of the three subfamilies. Thus 6-hydroxyflavones were found in both the Pitcairnioideae (in 50%) and the Tillandsioideae (in 14%) but patuletin (in 19%), gossypetin (in 1 species) and methylated 6-hydroxymyricetin derivatives (in 24%) were detected only in the Tillandsioideae. A new flavonol, 6,3′,5′-trimethoxy-3,5,7-4′-tetrahydroxyflavone, was identified as the 3-glucoside in Tillandsia usneoides and a new glycoside, patuletin 3-rhamnoside, in Vriesea regina. Myricetin glycosides were found only in the Bromelioideae and their presence here and the concomitant absence of 6-hydroxyflavonoids could indicate the primitive condition of this subfamily. The flavonoid results, in toto, confirm the view based on morphology, that the Bromeliaceae occupies an isolated position in relation to other monocot families.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

The photochemical and fluorescence properties of whole cells,spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum

The photochemical activities and fluorescence properties of cells, spheroplasts and spheroplast particles from the blue-green alga Phormidium luridum were compared. The photochemical activities were measured in a whole range of wavelengths and expressed as quantum yield spectra (quantum yield vs. wavelength). The following reactions were measured: Photosynthesis (O₂ evolution) in whole cells; Hill reaction (O₂ evolution) with Fe(CN)₆^3? and NADP as electron acceptors (Photosystem II and Photosystem II+Photosystem I reactions); electron transfer from reduced 2,6-dichlorophenolindophenol to diquat (Photosystem I reaction). The fluorescence properties were emission spectra, quantum yield spectra and the induction pattern.On the basis of comparison between the quantum yield spectra and the pigments compositions the relative contribution of each pigment to each photosystem was estimated. In normal cells and spheroplasts it was found that Photosystem I (Photosystem II) contains about 90 % (10 %) of the chlorophyll a, 90 % (10 %) of the carotenoids and 15 % (85 %) of the phycocyanin. In spheroplast particles there is a reorganization of the pigments: they loose a certain fraction (about half) of the phycocyanin but the remaining phycocyanin attaches itself exclusively to Photosystem I (!). This is reflected by the loss of Photosystem II activity, a flat quantum yield vs. wavelength dependence and a loss of the fluorescence induction.The fluorescence quantum yield spectra conform qualitatively to the above conclusion. More quantitative estimation shows that only a fraction (20–40 %) of the chlorophyll of Photosystem II is fluorescent. Total emission spectrum and the ratio of variable to constant fluorescence are in agreement with this conclusion.The fluorescence emission spectrum shows characteristic differences between the constant and variable components. The variable fluorescence comes exclusively from chlorophyll a; the constant fluorescence is contributed, in addition to chlorophyll a, by phycocyanine and an unidentified long wavelength component.The variable fluorescence does not change in the transition from whole cells to spheroplasts. However, the constant fluorescence increases considerably. This indicates the release of a small fraction of pigments from the photosynthetic photochemical apparatus which then become fluorescent.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.