The study of the primary photoprocesses in Photosystem I of chloroplasts Recombination luminescence,chlorophyll triplet state and triplet-triplet annihilation

The dependence of the delayed luminescence of Photosystem I on the state of the reaction centers has been studied. Light flash induces a charge separation in the centers: $\text{P-700 · P-430}\textcolor{red}{}\text{P-700}{}^{\mathrm{+}}\text{· P-430}{}^{\mathrm{?}}$. Dark recombination of charges is accompanied by the recombination luminescence with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 20}\text{ms}$.If the centers are in the P-700 · P-430^? state or if P-430 is inactivated by heat, then flashing of Photosystem I generates the triplet state chlorophyll with $\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 0.5}\text{ms}$. The triplet state has been measured by the delayed fluorescence of chlorophyll at 20 °C and 77 °K and by the chlorophyll phosphorescence at 77 °K. The delayed fluorescence at 20 °C arises from the thermal activation of the triplet state up to the excited singlet level of chlorophyll and at 77 °K it is due to triplet-triplet annihilation. The quantum yield of the triplet formation, estimated by a comparison of the light saturation curves of delayed fluorescence at 20 °C and of P-700 photooxidation under the same experimental (optical) conditions, is ≈ 0.9 of the P-700⁺ yield. Only one triplet of chlorophyll can be generated per P-700. Under heat inactivation of P-430 the triplet formation is not observed when P-700 is oxidized.It is assumed that the triplet-triplet annihilation at 77 °K is related with the strong interaction between the chlorophyll molecules in the pigment complex of Photosystem I. The possibility of a triplet participation in the primary processes of photosynthesis is discussed.     

Magnetophotoselection applied to the triplet state observed by EPR in photosynthetic bacteria.

Magnetophotoselection (MPS) techniques have been used to study the triplet state observed by electron paramagnetic resonance (epr) in photosynthetic bacteria. Intact $\text{R.}\text{rubrum}$ chromatophores and systems in which the effects of energy transfer via antenna chlorophyll molecules have been minimized were examined. These preliminary results indicate that there is order in the bacteriochlorophyll antenna system, that the optical transition at 890 nm appears to be along the triplet y axis of the bacteriochlorophyll special pair (Bchl_sp), and that the resultant transition moment associated with 800 nm is approximately parallel to the long wavelength transition moment of the Bchl_sp.     

Triplet states in photosynthesis

A comparison of zero field splitting (ZFS) and spin polarization of triplet spectra of bacteriochlorophyll $\text{a in vitro}$ and $\text{in vivo}$ provides support for the special pair model of photoreactive chlorophyll in photosynthetic bacteria. Spin polarization of the triplet spectra is a new and unique probe of primary events in the light conversion act in photosynthesis.     

Photobiochemistry in the dark: photohemolysis of red cells sensitized by chlorpromazine-bioenergized triplet acetone system

Chlorpromazine is decomposed when it is treated with bioenergized triplet acetone from the 2-methylpropanal/red cells/O₂ system, forming chlorpromazine-5-oxide, with a concomitant strong hemolytic effect observed by a spectrophotometric method. Experiments with external superoxide dismutase, catalase, benzoate and bicarbonate indicate the absence of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$, H₂O₂ and OH· species as the precursor of the hemolytic effect.Comparison between the 2-methylpropanal/peroxidase/O₂ system and the 2-methylpropanal/red cells/O₂ system in the presence of chlorpromazine, indicate that essentially the same type of mechanism occurs in both cases.These results could explain the $\text{in}$$\text{vivo}$ hemolytic and toxic effect of chlorpromazine in the dark.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Zero field resonance and spin alignment of the triplet state of chloroplasts at 2°K

Microwave induced transitions in zero magnetic field have been observed in the photoinduced triplet of chloroplasts treated with dithionite by monitoring changes in the intensity of the 735 nm fluorescence band at 2°K. Similar results were obtained with chloroplasts treated with hydroxylamine plus 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination. The zero field parameters are $\text{D = 0.02794 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, $\text{E = 0.00382 ± 0.00007}\text{cm}{}^{\mathrm{?1}}$, i.e. equal to those of monomeric chlorophyll $\text{a}$ to within the experimental error. The photoinduced triplet appears to be linked to Photosystem II. This indicates that the low temperature 735 nm fluorescence band of chloroplasts is at least partly due to Photosystem II.     

The triplet exciton of chlorophyll a and carotenoid in solution and in photosynthetic antenna proteins

We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll $\text{a}\text{b}$ protein complex by high-intensity laser excitation. The carotenoid triplet (³Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of ³Car in CP-II decreased 11-fold as intensity was increased from 10¹⁶ to 2 · 10¹⁷ photons/cm² per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 10¹⁶ photons/cm² per s, the ³Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between ³Car and chlorophyll triplets which is compatible with the observed increased rate of ³Car decay. Indirect evidence suggests similar effects occur in LHC.     

Photoreduction of membrane bound cytochrome C by excited-state phenothiazine.

Ferricytochrome $\text{c}$ can be reduced in a photochemical reaction by excited state phenothiazine. This reaction is observed between phenothiazine which is solubilized by phospholipid artificial membranes and cytochrome $\text{c}$ which is adsorbed to the membrane surface. Under conditions when cytochrome $\text{c}$ is not bound to the phospholipid, the rate of reduction by phenothiazine is greatly reduced. The phosphorescence of phenothiazine is quenched in the presence of cytochrome $\text{c}$, implying that the excited triplet state interacts with cytochrome $\text{c}$. Oxygen inhibits the reaction since possibly, as a paramagnetic species, it increases intersystem crossing of the excited states of phenothiazine. On the basis of molecular models the proximity between the iron of ferricytochrome $\text{c}$ and phenothiazine is estimated to be over 20 Å.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Detection of triplet states in algae by zero-field resonance.

Triplet states have been detected and characterized by zero-field splitting parameters in $\text{Anacystis nidulans}$, $\text{Euglena gracilis}$, $\text{Porphyridium cruentum}$, and $\text{Chlorella vulgaris}$, using fluorescence-detected magnetic resonance in zero-field at 4.2 K. Monitoring the 720 nm photosystem I emission, transitions between triplet spin levels have been assigned to antenna chlorophyll of one of both photosystems; photochemical reactions of chlorophyll are observed in the presence of an inhibitor and strong light, probably resulting in photoreduction and pheophytinization.     

Controlled alkaline hydrolysis of steroidal α-bromoketones: New conditions and synthesis of 2α-hydroxy-3-ones

Controlled alkaline hydrolysis of 16α-bromo-17-keto steroids $\text{1}$, $\text{5}$ and $\text{7}$ with potassium carbonate and tetra-n-butylammonium hydroxide (n-Bu₄NOH) and synthesis of 2α-hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ by the controlled hydrolysis of the corresponding 2α-bromo-3-ones $\text{9}$, $\text{12}$ and $\text{15}$ are described. Treatm carbonate in aqueous acetone or with n-Bu₄NOH in aqueous dimethylformamide (DMF) gave 16α-hydroxy-17-ones $\text{3}$, $\text{6}$ and $\text{8}$ in 85–90% yield, respectively. 2α-Hydroxy-3-ones $\text{11}$, $\text{13}$ and $\text{16}$ were obtained by hydrolysis of the corresponding bromoketones $\text{9}$, $\text{12}$ and $\text{15}$ in high yields using the above conditions or sodium hydroxide in pyridine or DMF, respectively. Deuterium labeling experiments suggested that equilibration between the 2α-bromoketone $\text{9}$ and the 2β-bromo isomer $\text{10}$ precedes the formation of the ketol $\text{11}$ in which the true intermediate might be the 2β-isomer $\text{10}$. However, rearranged androstane derivatives, 3β-hydroxy-2-ones $\text{18}$ and $\text{20}$, were stereoselectively obtained by treatment of the bromoketones $\text{12}$ and $\text{15}$ with an excess amount of sodium hydroxide.     

Microdetermination of norepinephrine, 3,4-dihydroxyphenylethylamine, and 5-hydroxyyptamine from single extracts of specific rat brain areas

The conditions reported by Toru and Aprison (4) for extracting ACh in specific brain areas were tested to determine whether 5-HT, NE, and dopamine were also extracted quantitatively. It was found that the extraction solution used in brain ACh determinations, 15% 1N formic acid plus 85% acetone ($\text{v}\text{v}$), was also excellent for extraction of NE, 5-HT, and dopamine from different brain areas. Experimental conditions are given for the microdetermination of all three biogenic amines in such a single extract of a specific rat brain area. The methods are based on previously published fluorometric methods; these have been scaled down or modified slightly to permit analyses of small aliquots. The concentration of 5-HT, NE, and dopamine in the telencephalon, diencephalon plus mesencephalon, pons plus medulla oblongata, and cerebellum of the rat are also reported using the described micromethods after extraction with 15% 1 N formic acid plus 85% acetone ($\text{v}\text{v}$).     

Requirement of a soluble protein for maximal activity of the mono-oxidase system of hepatic microsomes

Hepatic microsomes were shown to possess only about $\text{1}\text{3}$ to $\text{1}\text{2}$ of the aminopyrine and ethylmorphine (EM) N-demethylase activities of the 9000 × g supernatant fraction from which they were derived. Activity was restored with the 105,000 × g supernatant fraction (SF). The factor in SF is heat labile, precipitated by ammonium sulfate or acetone, none-dializable, and resists sedimentation at 170,000 × g for 4 hr. SF elevated the K_m for EM N-demethylation. These observations suggest that the factor in SF is a macromolecule which functions as an unknown component of the monoxidase system, as an activator of the system, or by removing an inhibitor of the system.     

Formation of aqueous pores in the human erythrocyte membrane after oxidative cross-linking of spectrin by diamide

Oxidation of erythrocyte membrane SH-groups by diamide and tetrathionate induces cross-linking of spectrin (Haest, C.W.M., Kamp, D., Plasa, G. and Deuticke, B. (1977) Biochim. Biophys. Acta 469, 226–230). This cross-linking was now shown to go along with a concentration- and time-dependent enhancement of membrane permeability for hydrophilic nonelectrolytes and ions. The enhancement is specific for oxidative SH-group modifications, is reversible by reduction of the induced disulfides, can be suppressed by a very brief pre-treatment of the cells with low concentrations of $\text{N-}\text{ethylmaleimide}$ and is strongly temperature-dependent. The pathway of the induced permeability discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}\text{3–8}\text{kcal/mol}$). These findings are reconcilable with the formation of a somewhat inhomogeneous population of aqueous pores with radii probably $\text{? 0.65}\text{nm}$. Estimated pore numbers vary with the size of the probe molecule. Assuming a diffusion coefficient as in bulk water within the pore, at least 20 pores per cell have to be postulated; more realistic lower diffusion coefficients increase that number. Alterations of the lipid domain by changes of cholesterol contents and insertion of hexanol or nonionic detergents alter the number or size of the pores. Since aggregation of skeletal and intrinsic membrane proteins also occurs after the SH-oxidation, in parallel to the formation of membrane leaks, one may consider (a) defects in the disturbed bilayer interface, (b) a mismatch between lipid and intrinsic proteins or (c) channels inbetween aggregated intrinsic proteins as structures forming the pores induced by diamide treatment.     

Purification of the prothoracicotropic hormone from the tobacco hornworm Manducasexta

Standard biochemical procedures were used to purify the prothoracicotropic hormone (PTTH) 4400 fold from whole head extracts of $\text{Mandura}$$\text{sexta}$ fifth instar larvae. Hormonal activity was bioassayed by injection into neck-ligated fourth instar larvae. The hormone was stable to heating at 85°C. Ammonium sulfate and acetone fractionation provided a crude preparation which showed dose-dependent activity in the bioassay. Chromatography on Sephadex G-100, DEAE-Sephadex, and hydroxylapatite gave a preparation with 2.6 $\text{Manduca}$ PTTH units/μg protein (4400-fold purification). Activity was sensitive to proteolytic enzymes. Further purification by preparative electrophoresis gave a preparation which migrated as a single band in two polyacrylamide gel electrophoresis systems. A molecular weight estimate of 25,000 Daltons was obtained for this bands on SDS polyacrylamide gels.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Isolation and primary structure of endorphin from salmon pituitary glands.

Endorphin has been isolated from an acid acetone extract of the pituitary of the salmon $\text{Oncorhynchus}\text{keta}$ by ion exchange chromatography and gel filtration. Sequence analysis revealed it to be a nonacosapeptide with following primary structure: Ac-Tyr-Gly-Gly-Phe-Met-Lys-Pro-Tyr-Thr-Lys-Gln-Ser-His-Lys-Pro-Leu-Ile-Thr-Leu-Leu-Lys-His- Ile-Thr-Leu-Lys-Asn-Glu-Gln-OH. It appears that the amino terminal segment which is necessary for analgesic activity is conserved through the evolution of vertebrate except for the blocking of the amino terminal of salmon endorphin.     

Structure of rhodotorucine A, a novel lipopeptide,inducing mating tube formation in Rhodosporidiumtoruloides

Rhodotorucine $\text{A}$ is a peptidyl factor which induces mating tube formation in $\text{Rhodosporidium}\text{toruloides}$. The amino acid sequence of the factor was determined by Edman degradation and enzymatic hydrolysis. Rhodotorucine $\text{A}$ was shown to contain a lipophilic amino acid, S-farnesyl cysteine, at C-terminus by proton magnetic resonance, mass spectrometry and chemical synthesis. We proposed the following structure for rhodotorucine $\text{A}$. H-Tyr-Pro-Glu-Ile-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-farnesyl)-OH     

Study of the interaction of polynucleotide chains with oligomers by means of chromatography. II. Effect of magnesium ions and noncomplementary bases on the stability of complexes

The interaction of the oligonucleotides ApA, ApApA, ApApC and ApApU with poly(U) and (Ip)₅I and (Ip)₆ with poly(C) has been studied by means of equilibrium gelfiltration through Sephadex.From sorption isotherms the free energies, energies and entropies of complexing have been computed for different concentrations of magnesium ions in the medium.The stoichiometric ratio of polymers to oligomers has been measured and found equal to 2 in the case of ApApA and ApApC. This shows that the cytidylic acid residue is included in the ternary complex. But in the case of ApApU the noncomplementary base is partly squeezed out of the complex.The stacking free energy of neighbouring oligomers has been found to be in the range 1000–3000 $\text{cal}\text{mole}$ depending on the conditions.The stoichimetric ratio has been found to be 1 in the case of poly(C): oligo(I), the stacking energy is equal to 1.2 $\text{kcal}\text{mole}$. The effect of magnesium is somewhat different in the case of double and triple helices and probably reflects the formation of coordination compounds with the nitrogen bases of nucleotides.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.