In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

Translational inhibition by heme of the synthesis of hepatic δ-aminolevulinate synthase in a cell-free system

Synthesis of δ-aminolevulinate synthase in a rabbit reticulocyte lysate system directed by total polysomes from the liver of allylisopropylacetamide-treated rats was studied with the combined use of [³H]leucine and a specific rabbit antibody. The protein synthesis observed in the cell-free system employed represented mainly the peptide chain elongation and its termination rather than the net synthesis involving initiation. Synthesis of δ-aminolevulinate synthase in this cell-free system was inhibited progressively with the increased addition of hemin; the synthesis was reduced to about 40% by about 30 μM hemin. Synthesis of total protein, however, was not significantly affected by the addition of hemin. The data obtained suggest that heme inhibits a peptide chain elongation step in the synthesis of δ-aminolevulinate synthase.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

New chemical inducers of carotenoid biosynthesis

Five compounds having a striking effect on the color of Marsh seedless grapefruit are reported. The compounds, namely [β-(diethylamino)-ethoxy]-benzene, [γ-(diethylamino)-propoxy]-benzene, [δ-(diethylamino)-butoxy]-benzene, 4-[β-(diethylamino)-ethoxy]-benzaldehyde, and diethylaminoethyl anisolate caused a 5- to 12-fold increase in the carotene content. Lycopene, not normally accumulated, became the major pigment. The mode of action appears to be similar to that of CPTA.     

Effect of 2,2′-bipyridyl on porphyrin synthesis in etiolated and light-treated barley leaves

The effects of 2,2′-bipyridyl on porphyrin formation differed in illuminated and dark-treated barley leaves. In the dark, bipyridyl treatment increased photoconvertible protochlorophyllide (Pchlide, P650) and decreased the protohaem content. The increase in Pchlide could not be wholly accounted for by a diversion of ‘substrate’ from protohaem synthesis. The rate of Pchlide regeneration was slightly higher in chelator treated leaves which suggests increased δ-aminolaevulinic acid (ALA) synthesis. Only small quantities of Mg-protoporphyrinmonomethylester (Mg-protoME) were detected in etiolated leaves treated with bipyridyl in the dark. Protochlorophyll (P630) synthesis from exogenously supplied ALA was lower in the chelator treatments. The results suggest that only when substantial quantities of ALA are being utilized in dark-grown leaves does a ‘metal’ become limiting in the bipyridyl treated leaves. In the light, bipyridyl inhibited chlorophyll synthesis, again suggesting that when substantial amounts of ALA were being utilized a ‘metal’ becomes rate limiting. Bipyridyl treatment also inhibited ALA production in light-treated leaves. The incorporation of glycine-[¹⁴C] into ALA in the presence of bipyridyl was severely restricted compared to the incorporation of glutamate-[¹⁴C]. The data suggest two pathways for ALA synthesis; the classical ALA-synthetase which utilizes glycine and is operative in dark-grown leaves and a second enzyme system, which uses glutamate, and is of quantitative importance in the light.     

Paleosalinity in a brackish lake during the Holocene based on stable oxygen and carbon isotopes of shell carbonate in Nakaumi Lagoon, southwest Japan

Based on the relationship between salinity and δ¹⁸O and δ¹³C of modern shells in the Lake Nakaumi-Shinji lagoon system (southwestern Japan), where the salinity changes regularly from ca. 1 PSU to 34 PSU, a paleosalinity record for Nakaumi Lagoon during the Holocene has been derived from bulk mollusk shell δ¹⁸O and δ¹³C data. The robust relationships between the salinity and modern shell δ¹⁸O_ar and δ¹³C_ar (aragonite) were used to calibrate the paleosalinity reconstruction. The salinity relationships are expressed by the regressions:

Salinity (PSU)=3.86 δ¹⁸O_ar(‰ VPDB)+33.9 (n=18, r=0.978)     

The bortezomib-induced mitochondrial damage is mediated by accumulation of active protein kinase C-delta

Bortezomib (PS-341) is an inhibitor of the S26 proteasome. Bortezomib induces mitochondrial damage but the exact mechanism remains unclear. We studied PKC-delta, a kinase that is regulated by proteasome degradation and translocates to mitochondria in apoptosis, and examined whether PKC-delta could be a potential mediator of bortezomib-induced mitochondrial damage. Co-incubation of bortezomib with a PKC-delta inhibitor, rottlerin, suppressed bortezomib-induced apoptosis in U937 cells. Western analysis of U937 cells treated with bortezomib revealed accumulation of full-length PKC-delta in the first 4 h. By 16 h an active catalytic fragment of PKC-delta accumulated in mitochondria. The cleavage of PKC-delta after bortezomib treatment was mediated by caspases, because a pan-caspase inhibitor BAF prevented the appearance of the active fragment of PKC-delta. These findings indicate that accumulation of the active PKC-delta fragment in mitochondria is responsible for bortezomib-induced mitochondrial damage.     

Carbon and hydrogen isotopic fractionation during lipid biosynthesis in a higher plant (Cryptomeria japonica)

Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol), extracted from Cryptomeria japonica leaves, were determined in order to understand isotopic fractionations occurring during lipid biosynthesis in this species. All lipid biomolecules were depleted in both ¹³C and D relative to bulk tissue and ambient water, respectively. n-Alkyl lipids associated with the acetogenic pathway were depleted in ¹³C relative to bulk tissue by 2.4-9.9‰ and depleted in D relative to ambient water by 91-152‰. C₁₅- and C₃₀-isoprenoid lipids (sesquiterpenes, squalene and β-sitosterol) associated with the mevalonic-acid pathway are depleted in ¹³C relative to bulk tissue by 1.7-3.1‰ and depleted in D relative to ambient water by 212-238‰. C₂₀-isoprenoid lipids (phytol and diterpenoids) associated with the non-mevalonic-acid pathway were depleted in ¹³C relative to bulk tissue by 4.6-5.9‰ and depleted in D relative to ambient water by 238-303‰. Phytol was significantly depleted in D by amounts up to 65‰ relative to other C₂₀ isoprenoid lipids. The acetogenic, mevalonic-acid and non-mevalonic-acid pathways were clearly discriminated using a cross-plot between the carbon and hydrogen isotopic fractionations.     

Hydrogen, carbon and nitrogen isotopic fractionations during chlorophyll biosynthesis in C3 higher plants

We determined hydrogen, carbon and nitrogen isotopic compositions of chlorophylls a and b isolated from leaves of five C3 higher plant species (Benthamidia japonica, Prunus japonica, Acer carpinifolium, Acer argutum and Querus mongloica), and hydrogen and carbon isotopic compositions of phytol and chlorophyllides in the chlorophylls to understand isotopic fractionations associated with chlorophyll biosynthesis in these species. Chlorophylls are depleted in D relative to ambient water by approximately 189 per thousand and enriched in (13)C relative to bulk tissue by approximately 1.6 per thousand. These data can be explained by the contribution of isotopic fractionations during phytol and chlorophyllide biosyntheses. Phytol is more depleted in both D (by approximately 308 per thousand) and (13)C (by approximately 4.3 per thousand), while chlorophyllides are less depleted in D (by approximately 44 per thousand) and enriched in (13)C (by approximately 4.8 per thousand). Such inhomogeneous distribution of isotopes in chlorophylls suggests that (1) the phytol in chlorophylls reflects strong D- and (13)C-depletions due to the isotopic fractionations during the methylerythritol phosphate pathway followed by hydrogenation, and (2) the chlorophyllides reflect D- and (13)C-enrichments in tricarboxylic acid cycle. On the other hand, chlorophylls are slightly ( approximately 1.2 per thousand) depleted in (15)N relative to the bulk tissue, indicating that net isotopic fractionation of nitrogen during chlorophyll biosynthesis is small compared with those of hydrogen and carbon.