Water-soluble chlorophyll protein of Brassica oleracea var. Botrys (cauliflower)

A water-soluble chlorophyll protein was prepared from Brassica oleracea var. Botrys (cauliflower) and purified by (NH₄)₂SO₄ fractionation and by chromatography on a DEAE-cellulose column. The chlorophyll protein contained chlorophylls a and b in the ratio 6:1, and no carotenoids. The molecular weight, determined by means of gel filtration on Sephadex G-100, was 78000. The chlorophyll protein showed absorption peaks at 273, 340, 384, 420, 438, 465, 628, 674 and 700 nm. Since the three bands at 384, 420 and 438 nm all have approximately the same height, the spectrum is different from that of chlorophyll a in organic solvents. The fluorescence of the chlorophyll protein showed a peak at 683 nm, with shoulders at 706 and 745 nm at room temperature, and peaks at 685, 706 and 744 nm at the temperature of liquid N₂. An apo-protein was prepared by removing the chlorophylls with 2-butanone and purified by precipitation with (NH₄)₂SO₄. The apo-protein thus prepared had an absorption band at 273 nm but none at longer wavelengths. The apo-protein could be combined with chlorophylls, forming a chlorophyll protein which had spectral characteristics similar to those of the original.     

The yolk polypeptides of a free-living rhabditid nematode

• 1.1. The yolk proteins of hermaphrodite Dolichorhabditis sp. (Nematode, Rhabditida) are composed of at least three polypeptides: VT1, VT2 and VT3 with molecular masses of 175.2, 107 and 82 kDa respectively.
• 2.2. All three yolk polypeptides make up at least one native protein complex which can be resolved by PAGE.
• 3.3. The yolk proteins are glycosylated and can be isolated by chromatography in Con A-Sepharose.
• 4.4. Partial chymotryptic hydrolysis shows that VT2 in different from its C. elegans homologue, YP115.
• 5.5. The main polypeptides synthesized by whole animals are the yolk components which are actively secreted in the incubation medium.

     

Behavior of silkworm yolk protein on phospholipid membranes

During early development, the plasma membrane of silkworm (Bombyx mori) eggs undergoes a superficial cleavage that separates the blastodermal protoplasm and the yolk. To test whether the blastoderm absorbs yolk through the plasma membrane in B. mori, we studied the interaction of phospholipid membranes and yolk using a phospholipid planar bilayer membrane (PBM) and liposomes. In addition, egg-specific protein (ESP; 225 kDa), a yolk protein that is specific to B. mori eggs, was collected by fractionating the eggs. Liposomes were mixed with either B. mori yolk or ESP, and observed under an electron microscope. This showed that the phospholipid membrane was spanned by fine particles 10-20 nm in diameter. Both yolk and ESP caused the PBM to become extraordinarily leaky, with a membrane potential of −70 mV for yolk and −198 mV for ESP. These results suggest that although it is a water-soluble protein, ESP permeates the phospholipid membrane without the help of enzymes.     

Ecdysteroids and oocyte development in the black fly <Emphasis Type="Italic">Simulium vittatum</Emphasis>

Background

Oocyte development was studied in the autogenous black fly, Simulium vittatum (Diptera, Nematocera), a vector of Onchocerca volvulus, the causative agent of onchocerciasis.

Results

Oocyte growth was nearly linear between adult eclosion and was complete by 72 hours at 21°C. The oocyte became opaque at 14 hours after eclosion indicating the initiation of protein yolk deposition. The accumulation of vitellogenin was measured using SDS-PAGE. The density of the yolk protein bands at about 200 and 65 kDa increased during the first and second days after eclosion. The amount of protein in the 200 kDa band of vitellogenin, determined using densitometry, rapidly increased between 12 and 25 hours after eclosion. Ecdysteroid levels were measured using a competitive ELISA. Ecdysteroid levels increased rapidly and subsequently declined during the first day after eclosion.

Conclusion

These data show a correlation between the appearance of vitellogenin in the oocyte, and the rise in ecdysteroids. A possible relationship to molting of the nematode, Onchocerca volvulus, is discussed.

     

Comparative vitellin pattern and cladogenesis in Bacillus (insecta: Phasmatodea)

• 1.1. Under denaturing conditions (SDS-PAGE) the two natural vitellins of Bacillus taxa released five different polypeptides (A₁, A₂, A₃, B₁, B₂).
• 2.2. A₂ and B₂ bands from the two bisexual species (B. rossius and B. grandii) were found to differ; furthermore a non-vitellin yolk protein characterizes the subsepecies B.g. benazzii.
• 3.3. From gels and their densitometric scanning profiles it is clear that parental polypeptides are expressed in the thelytokous parthenogenetic hybrids (B. whitei, B. lynceorum) and in the hybridogenetic B. rossius-grandii benazzii.
• 4.4. A comparative approach of vitellin patterns appears fully adequate for tracing phylogenetic relationships and recognizing cladogenetic events.

     

Orange protein from <Emphasis Type="Italic">Desulfovibrio alaskensis</Emphasis> G20: insights into the Mo–Cu cluster protein-assisted synthesis

A novel metalloprotein containing a unique [S₂MoS₂CuS₂MoS₂]^3? cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI–MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S–Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV–visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Isolation of crustacean egg yolk lipoproteins by differential density gradient ultracentrifugation

• 1.1. Egg yolk lipoproteins from four species of Crustacea were isolated by differential density gradient ultracentrifugation.
• 2.2. Egg yolk proteins from freshwater prawn, striped stone crab and mitten crab consissted of high-density lipoprotein (HDL) and lipid-free protein, while low-density lipoprotein (LDL) was present in the egg yolk protein of sand crayfish as well as HDL and lipid-free protein.
• 3.3. HDL was a major component in the egg yolk proteins from four species of Crustacea. HDL was identical to egg yolk lipovitellin.
• 4.4. Both HDL and LDL possessed phospholipid as a major lipid.
• 5.5. HDL, but not LDL, contained carotenoids. The color of HDL from mitten crab showed a reddish purple and was distinct from other Crustacea whose color was orange. The reddish purple color was characterized by an absorption flexion at 600–650 nm.

     

Substrate and inhibitor complexes of dihydrofolate reductase from amethopterin-resistant L1210 cells.

Dihydrofolate reductase (EC 1.5.1.3), purified to homogeneity from an amethopterin-resistant subline (R6) of cultured L1210 murine leukemia cells, has been used to study enzyme-substrate and enzyme-inhibitor complexes. NADPH, NADP⁺acid-modified NADPH (λ_max at 265 nm, elevated absorbance at 290 nm), 2′-phosphoadenosine-5′-diphosphate ribose, dihydrofolate, and amethopterin formed binary complexes with the enzyme. Ternary complexes could be formed by admixing the enzyme with: (a) NADPH and amethopterin; (b) NADP⁺ and tetahydrofolate; and (c) acid-modified NADPH and dihydrofolate. All of these complexes migrated as stable well-defined bands on polyacrylamide gel electrophoresis at pH 8.3. The bands could be visualized by staining both for enzyme activity and for protein. These binary and ternary complexes were also stable to extensive dialysis. Spectra of the dialyzed enzyme complexes indicated that each ligand was present at an equimolar ratio with the enzyme.     

Cyclam cored luminescent dendrimers as ligands for Co(II), Ni(II) and Cu(II) ions

We have investigated the photophysical properties of two dendrimers containing a cyclam core decorated with 4 naphthyl units (G0), 12 dimethoxybenzene and 16 naphthyl units (G2). These dendrimers show fluorescence bands that can be assigned to naphthyl localized excited states (λ_max = 337 nm), naphthyl-amine exciplexes (λ_max = 470 nm) and, for G2, naphthyl excimers (λ_max ca. 400 nm). Cyclam is a very good ligand for transition metal ions and we have investigated complex formation between these dendritic ligands and Ni(II), Co(II) and Cu(II), added as nitrate salts. This process can be monitored by the strong changes, both in shape and intensity, observed in the emission spectra of these dendrimers. Complexation with Cu(II) causes not only changes in the relative intensities of the fluorescence bands, but also the appearance of a new absorption band in the near UV spectral region. An analysis of the titration curves has allowed us to obtain clear evidence for the formation of not only 1:1 species, but also 1:2 metal to ligand species. G2 shows a clear preference, compared to G0, in forming complexes with a 1:2 metal-to-ligand stoichiometry, although it possesses very bulky dendrons appended to the cyclam central unit.     

Excitation spectra of chlorophyll fluorescence in spinach and barley chloroplasts at 4 K

Excitation spectra of chlorophyll a fluorescence in chloroplasts from spinach and barley were measured at 4.2 K. The spectra showed about the same resolution as the corresponding absorption spectra. Excitation spectra for long-wave chlorophyll a emission (738 or 733 nm) indicate that the main absorption maximum of the photosystem (PS) I complex is at 680 nm, with minor bands at longer wavelengths. From the corresponding excitation spectra it was concluded that the emission bands at 686 and 695 nm both originate from the PS II complex. The main absorption bands of this complex were at 676 and 684 nm. The PS I and PS II excitation spectra both showed a contribution by the light-harvesting chlorophyll $\text{a}\text{b}$ protein(s), but direct energy transfer from PS II to PS I was not observed at 4 K. Omission of Mg²⁺ from the suspension favored energy transfer from the light-harvesting protein to PS I. Excitation spectra of a chlorophyll b-less mutant of barley showed an average efficiency of 50–60% for energy transfer from β-carotene to chlorophyll a in the PS I and in the PS II complexes.     

Characterization of cryptomonad phycoerythrin and phycocyanin

Phycoerythrin, a chromoprotein, from the cryptomonad alga Rhodomonas lens is composed of two pairs of nonidentical polypeptides (α₂β₂). This structure is indicated by a molecular weight of 54,300, calculated from osmotic pressure measurements and by sodium dodecyl sulfate (SDS) gel electrophoresis, which showed bands with molecular weights of 9800 and 17,700 in a 1:1 molar ratio. The s_20,w⁰ of 4.3S is consistent with a protein of this molecular weight. Similar results were obtained with another cryptomonad phycoerythrin and a cryptomonad phycocyanin. Electrophoresis after partial cross-linking by dimethyl suberimidate revealed seven bands for the cryptomonad phycocyanin and six bands for cryptomonad phycoerythrin and confirmed the proposed structure. Spectroscopic studies on α and β subunits of cryptomonad phycocyanin and phycoerythrin were carried out on the separated bands in SDS gels. The individual polypeptides possessed a single absorption band with the following maxima: phycoerythrin (R. lens), α at 565 nm, β at 531 nm; phycocyanin (Chroomonas sp.), α at 644 nm, β at 566 nm. Fluorescence polarization was not constant across the visible absorption band regions of phycoerythrin (R. lens and C. ovata) with higher polarizations located at higher wavelengths, as had also been previously shown for cryptomonad phycocyanin (Chroomonas sp.). Combining the absorption spectra and the polarization results indicates that in each case the β subunit contains sensitizing chromophores and the α subunit fluorescing chromophores. The CD spectra of cryptomonad phycocyanin and both phycoerythrins were similar and were related to the spectra of the individual subunits. In Ouchterlony double-diffusion experiments the cryptomonad phycoerythrins and phycocyanins cross-reacted, with spurring, with phycoerythrin isolated from a red alga. The cryptomonad phycoerythrins were immunochemically very similar to each other and to cryptomonad phycocyanin, with little spurring detected.     

Characterization of PHB1 and Its Role in Mitochondrial Maturation and Yolk Platelet Degradation during Development of Artemia Embryos

Background

To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.

Principal Finding

Our study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.

Conclusions/Significance

PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Raman spectrum of protocatechuate dioxygenase from Pseudomonas putida. New low frequency bands.

The Raman spectrum (441.6 nm excitation) of protocatechuate 3,4-dioxygenase (PCD) from Pseudomonas putida shows resonance enhanced bands at 1605, 1504, 1270, 858, and 830 cm^?1 which are due to the p-hydroxyphenyl group of tyrosine coordinated to iron. In addition, we observe strong resonance enhanced bands at 592 and 524 cm^?1 and weak (presumably iron-ligand) vibrations at 465, 423, and 371 cm^?1. Recent publications of the Raman spectrum of PCD from Pseudomonas aeruginosa (Tatsuno $\text{et al}$, J. Am. Chem. Soc. 100, 4614–4615 (1978) and Keyes $\text{et al}$, Biochem. Biophys. Res. Comm. 83, 941–945 (1978) using 488 and 514 nm excitation did not report these bands. Our 441.6 nm excitation Raman spectrum of human serum transferrin, another metalloprotein with an iron-tyrosine linkage, does not show the 592 and 524 cm^?1 bands and has only two very weak bands at about 423 and 364 cm^?1. We discuss several interpretations of these data.     

Synthesis of vitellogenic and non-vitellogenic yolk proteins by the fat body and the ovary of Leptinotarsa decemlineata

• 1.1. Isolated ovaries of egg laying females synthesize and secrete three yolk proteins (two vitellogenins and chromoprotein 2).
• 2.2. The contribution of ovarian tissue to total yolk protein production is very small, the major site of synthesis of the three yolk proteins being the fat body.
• 3.3. There is a time lag between yolk protein synthesis by the fat body and yolk protein sequestration by the ovary.
• 4.4. In egg laying females, within 1 hr after the synthesis of both vitellogenins by the fat body, they appear in the oocytes as vitellins.

     

Renal cortical cells in primary monolayer culture. Enzymatic changes and morphological observations.

Stress fibers and bands of intermediate filaments (100 Å) were studied in cultured non-muscle cells using laser microbeam techniques. Wavelengths of 532, 537 and 280 nm were used, and no artificial chromophores were employed. Lesions were assayed using a combination of phase contrast, polarizing and transmission electron microscopy (TEM). (1) Stress fibers 1–2 μm in diameter were narrowed or completely servered by irradiation at 532, 537 and 280 nm. Stress fibers could be grouped into two classes: (a) those whose severed ends separated during the first few seconds following laser irradiation (46% of fibers irradiated); (b) those fibers which showed no movements (54%). Microtubules which paralleled stress fibers persisted in the presence of colcemid for up to 5 h, and alignment of the severed stress fiber ends was maintained even in their absence. Injured stress fibers appear to be repaired within 1 h of irradiation. (2) Bands of 100 Å filaments were induced in non-muscle cells in secondary cultures of neonatal rat heart by exposure to colcemid. Lesions which appeared as phase dense spots were induced in these bands by irradiation at 532, 537 and 280 nm. The positions of the lesions in the band relative to one another did not change over several hours despite movements of the entire band. These studies demonstrate that (a) stress fibers may be an excellent system in which to study subcellular repair; (b) induced bands of 100 Å filaments probably move passively in the cells containing them; (c) laser irradiation of cytoplasmic filaments in non-muscle cells does not require the introduction of an artificial chromophore.