Temperature-Induced Change of Chlorophyll a Forms in Phosphatidylcholine Liposomes

Absorption spectra of chlorophyll a in phosphatidylcholine liposomesat different temperatures were analyzed by a curve fitting method.The absorption spectrum was found to be composed of one majorband with a peak at 670–671 nm and minor bands with peaksat 650–652, 662–663 and 684–686 nm. Upon coolingbelow the phase transition temperature of the lipid, the componentabsorbing at 670–671 nm increased significantly at theexpense of the component absorbing at 662–663 nm. No changein the extents of other bands was observed. ¹ CIW-DPB Publication No. 795. ²On leave from the Department of Biology, Faculty of Science,Kanazawa University, Marunouchi, Kanazawa 920, Japan. (Received December 20, 1982; Accepted April 27, 1983)     

The Light Induced Protochlorophyll–Chlorophyll a-Transformation and the Succeeding Interconversions of the Different Forms of Chlorophyll

Curve resolution into Gaussian components of the absorption spectra during the varying stages of the Shibata shift in dark grown, irradiated leaves of barley indicates that the chlorophyll a forms formed after irradiation consist of the same main components which have been reported to be present in all hitherto investigated plant materials (peak values in the red region 662, 670, 677 and 683 nm, respectively) but in varying proportions. The spectra during the Shibata shift proper can be satisfied by a mixture of two single components gradually changing their proportions, although a four component system gives a still better fit to the measured absorption curves. It is also shown that curves taken before and after the shift and added together in the appropriate proportions will match the absorption spectrum measured with peak at the isosbestic point (after ca. 15 min at room temperature).     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m²) was investigated for the chlorophyll(ide) pigments formed after photoreduction of the protochlorophyllide in dark grown leaves of wheat. Connections were found between changes in absorption spectrum in vivo (the Shibata shift and the late red-shift) and changes in photostability both in young (five-day) and old (12-day) leaves. The photostability of both the 684-form and the 673-form as well as the rate of the changes in photostability (the Shibata shift and the late red-shift) decreased with the age of the dark grown plants. It was concluded that the more pronounced decrease in the chlorophyll(ide) contents found at irradiation of older dark grown leaves mostly depended on the lower rate of the changes in the photostability of the pigment in old leaves. No resynthesis of protochlorophyllide occurred before the onset of the late red-shift. The results and their connection with the lag in chlorophyll formation are discussed. This lag is more pronounced in older dark grown wheat.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The stability against high intensity irradiation (red light, 700 W m^?2) was investigated for the chlorophyll(ide) pigments formed after the primary photoreduction of the protochlorophyll(ide) in dark grown leaves of wheat. After photoreduction, most of the chlorophyll(ide) exists in a form with an absorption maximum at 684 nm. This form is gradually transformed into a form with an absorption maximum at 673 nm (the Shibata shift). It was possible to ascribe a specific photostability to each of the pigment forms. This photostability was higher for the 673-form than for the 684-form. A red-shift in the absorption maximum following upon the Shibata shift, reflects the successive transformation of the 673-form into other pigment forms, which were quite photostable at the intensity used.     

The Photostability of Different Chlorophyll Forms in Dark Grown Leaves of Wheat

The effect of denaturing treatments on the stability against high intensity irradiation (red light, 700 W m^?2) was investigated in vivo for various chlorophyll forms in wheat. Three pigment forms were investigated: the 650-form (protochlorophyllide) present in dark grown leaves; the 684-form (chlorophyllide) formed within 5 s after photoreduction of the 650-form; and the 673-form (chlorophyll), into which the 684-form has been transformed 25 min after photoreduction of the 650-form. (The pigment forms are denoted by their absorption maxima in the red region before denaturation.) Two denaturing treatments were used: heat treatment (water of 55°C for 2 min) and freezing and thawing (freezing in liquid nitrogen followed by thawing in water of 25°C). Heat treatment as well as freezing and thawing caused a shift in the absorption peak of the two nonesterified pigment forms. The peak of of the chlorophyllide 684-form shifted to 673 nm and that of the protochlorophyllide 650-form to 636 nm. The absorption maximum of the chlorophyll 673-form was not affected by the above treatments. Heat treatment as well as freezing and thawing had profound effects on the structural organization of the plastid pigments, as shown by a decrease in the photostability. For the 684-form, heat treatment reduced the photostability by a factor of about 14 (half-life in strong light changed from 170 s to 12 s). Freezing and thawing also reduced the photostability, although the effect was less pronounced (c. 3–4 times decrease in half-life). Upon transformation of the chlorophyllide 684-form into the chlorophyll 673-form (the Shibata-shift) the pigments became less sensitive to light, and were no longer “aggregated” by heat treatment. The “aggregating” effect of freezing and thawing was still present after the Shibata shift. The results thus verify a clear difference in structural organization of the 684-form and the 673-form, since the two pigment forms were differently affected by heat treatment. The 650-form behaved similarly to the 684-form, although it appeared to be slightly less aggregated by heat treatment. — The decrease in photostability, caused by heat treatment of the 684-form, changed the kinetics for the photodecomposition from a first towards a second order reaction.     

The Length of a Generation

After comparing a number of definitions of the length of a generation, two new ones are proposed. One is the mean age of mothers having new born children in the current population, the other is the period of decay of departures from the stable age distribution. Numerical examples are given.     

IS1549 from Mycobacterium smegmatis Forms Long Direct Repeats upon Insertion

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.     

Wave Forms of Caenorhabditis elegans in a Chemical Attractant and Repellent and in Thermal Gradients

The wave forms and activity patterns of Caenorhabditis elegans were examined on agar in the presence of known chemical attractants (NaCl) and repellents (D-tryptophan), and in thermal gradients. Total activity was reduced in both attractants and repellents. Different combinations of transfers between chemicals were investigated. Two thresholds were found for NaCl: 10^-3 M NaC1 caused reduced activity; 10⁻⁵ M NaCl increased reversals. D- or L-tryptophan influenced neither orientation nor the ability of thermally acclimatized individuals to remain at their eccritic temperature.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.     

The Light-harvesting Chlorophyll a/b-Protein Complex of Chlamydomonas reinhardii

The molecular organization of chlorophyll in Chlamydomonas reinhardii has been shown to be essentially similar to that in higher plants. Some 50% of the chlorophyll in Chlamydomonas reinhardii chloroplast membranes has been shown to be located in a chlorophyll a/b-protein complex. The complex was isolated in a homogeneous form by hydroxylapatite chromatography of sodium dodecyl sulfate extracts of the chloroplast membranes. Its absorption spectrum exhibits two maxima in the red region at 670 and 652 nm due to the presence of equimolar quantities of chlorophylls a and b in the complex. Preparations of the chlorophyll-protein also contain some of each of the carotenoids observed in the intact chloroplast membrane, but not in the same proportions. The native complex (S value = 2.3S) exhibits a molecular weight of 28,000 ± 2,000 on calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on the basis of its amino acid composition and other data a more probable molecular weight of about 35,000 was calculated. Each 35,000 dalton unit contains three chlorophyll a and three chlorophyll b molecules, and on the average one carotenoid molecule conjugated with probably a single polypeptide of 29,000 daltons. Comparison of spectral and biochemical characteristics demonstrates that this algal chlorophyll-protein is homologous to the previously described major light-harvesting chlorophyll a/b-protein of higher plants. It is anticipated that the Chlamydomonas complex functions solely in a light-harvesting capacity in analogy to the function determined for the higher plant component.     

Stacking Capacity and Chlorophyll Forms of Thylakoids in Normal and Mutant Maize Chloroplasts of Different Granum Content

Stacking of chloroplast lamellae, isolated from normal and carotenoid mutant chloroplasts of maize (Zea mays L.), was determined after a high-salt treatment. Stacking of isolated lamellae under favourable ionic conditions was almost identical with that occurring in intact chloroplasts; thus, differences in granum content could be attributed to the architectural properties of lamellae. Gaussian analyses, performed on the red band of room temperature absorption spectra, have shown that chloroplasts with lamellae of high stacking capacity contain relatively more Chl a₆₆₂ than chloroplasts containing lamellae of low stacking capacity. The presence of Chl a_705–708 was characteristic of preparations containing considerable amounts of stroma lamellae.     

Migration of Electronic Energy from Chlorophyll b to Chlorophyll a in Solutions

Absorption, emission, and fluorescence excitation spectra of pure solutions of chlorophyll a (Chl a) and chlorophyll b (Chl b) in diethyl ether and of equimolecular mixed solutions of the two pigments, were determined at room temperature as functions of concentration (in the range from 5 × 10^-6 M to 4 × 10^-3 M) and of wavelength of the exciting light (in the regions 380-465 and 550-650 nm). The efficiency of energy transfer from Chl b to Chl a, derived from these data, was found to depend on the wavelength of exciting light. Furthermore, the transfer efficiency calculated from sensitization of Chl a fluorescence by Chl b was substantially smaller than that calculated from quenching of Chl b fluorescence by Chl a. Both these effects are tentatively explained as evidence of superposition of a “fast” energy transfer (taking place before the Boltzmann distribution of vibrational energy had been reached) upon the “delayed” transfer, which takes place after vibrational equilibration. The first-named mechanism is made possible by overlapping of the absorption bands of the two pigments; the second, by overlapping of the emission band of Chl b and the absorption band of Chl a. The first mechanism can lead to repeated transfer of excitation energy between pigment molecules, the second only to a one-time transfer from the donor to the acceptor. Both mechanisms could be of the same, second-order type, with the transfer rate proportional to r^-6. An alternative is for the fast mechanism to be of the first order, with the transfer rate proportional to r^-3, but spectroscopic evidence seems to make this alternative less probable.     

油菜叶绿素b减少突变体Cr3529叶绿素生物合成的研究

利用吸收光谱和荧光光谱法测定了油菜叶绿素b减少突变体Cr3529子叶叶绿素生物合成途径中几种主要前体物质的含量.结果显示:突变体子叶中叶绿素生物合成第一个限速步骤的前体物质δ-氨基乙酰丙酸(ALA)含量与野生型油菜大致相同,饲喂ALA后的突变体及野生型油菜子叶中ALA含量均显著增加,但二者无显著差异;胆色素原含量在突变体中也未降低,而尿卟啉原Ⅲ含量仅为野生型的一半,粪卟啉原Ⅲ、原卟啉Ⅸ、镁原卟啉Ⅸ和原植基叶绿素的含量都明显低于野生型.结果证明,Cr3529突变体中叶绿素生物合成受阻于由胆色素原形成尿卟啉原Ⅲ的步骤,其叶绿素合成缺陷的机制和前体物质的累积与其它叶绿素b减少突变体明显不同.     

Influence of Culture Conditions on the Length of the Lag Period of Chlorophyll Synthesis in Preilluminated Dark-grown Euglena

Two parameters of the potentiation process, the lengths of preillumination and of the subsequent dark phase have been determined for Euglena cells which had been grown in the dark under two different culture conditions. In both cases a maximum potentiation response is obtained after a preillumination period of about 2 hours. The minimum length of the dark period necessary for maximum potentiation changes from 1 hour for cells which contain, at the moment of illumination, a high paramylum content, to 6 hours for cells which have a low paramylum content.     

叶绿素含量测定的简化

本文介绍一种避免称重、研磨、冲洗和过滤等繁琐步骤,仅将确定面积的叶片细丝用少量80%丙酮浸泡提取后便可比色计算的简便的测定叶绿素含量的方法。此法省时间、省溶剂,不受叶片含水量变化的干扰,适合于大批量叶片样品的测定,而且便于与以单位叶面积表示的光合速率联系起来分析实验结果。同时,指出一些可能影响测定结果的因素。     

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.