Subunit organization of Euglena chromatin

Digestion of $\text{Euglena}$ nuclei or extracted chromatin with micrococcal nuclease results in the identification of a repeating structure. The DNA repeat size, analyzed on agarose and polyacrylamide gels, is found to be 225±13 base pairs. DNase I digestion produces a serie of fragments multiples of roughly 10 bases. Eventhough pressure shearing is necessary to disrupt the though pellicule of the phytoflagellate, we confirm that, in $\text{Euglena}$, chromatin organization is similar to that of other eukaryotes.     

Light scattering,chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO₂ was added to a stream of CO₂-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf $\text{ATP}\text{ADP}$ ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, $\text{ATP}\text{ADP}$ ratios were maintained in the light. They decreased drastically only after darkening. Although $\text{ATP}\text{ADP}$ ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.     

Specificity of the serine protease inhibitor, phenylmethylsulfonyl fluoride.

Clarified cell-free extracts were prepared from rapidly dividing $\text{Bacillus subtilis}$ cells and from rabbit liver cells. These extracts were treated with [³H]-phenylmethylsulfonyl fluoride (PMSF) and analyzed by electrophoresis in isoelectric focusing polyacrylamide gels or detergent gels. Not less than 14 proteins in the $\text{B. subtilis}$ extracts and not less than 15 proteins in rabbit liver extracts reacted covalently with PMSF. These results suggest that PMSF is not as specific for serine proteases as sometimes supposed, and its effects in physiological experiments should be interpreted with caution.     

The detection of a bound ferredoxin in the photosynthetic lamellae of blue-green algae and other oxygen evolving photosynthetic organisms

The presence of an electron transport component with an EPR spectrum similar to that of a ferredoxin has been demonstrated in the blue-green alga $\text{Anabaena}$$\text{cylindrica}$, the green alga $\text{Euglena}$$\text{gracilis}$, and in chloroplasts from sorghum ($\text{Sorghum}$$\text{bicolour}$) and beans ($\text{Phaseolus}$$\text{vulgaris}$). The component is photoreduced at 77°K and is very similar to that previously reported in spinach. It seems likely that this component is a primary electron acceptor in photosynthesis in all of these organisms.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

Titles of related papers in other sections

Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ is closer to the in vivo form rather than the monomer.     

Resolution of the light-harvesting chlorophyll ab-protein of Vicia faba chloroplasts into two different chlorophyll-protein complexes

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll $\text{a}\text{b}$ ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll $\text{a}\text{b}$ ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.     

Partial purification of a chloroplast DNA polymerase from Euglena gracilis

A single DNA polymerase has been purified 965 fold from isolated chloroplasts of $\text{Euglena}$$\text{gracilis}$ with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.     

Adenosine 3',5' cyclic monophosphate in Euglena gracilis

$\text{Euglena gracilis}$ contains in high concentration the enzymes for the synthesis and degradation of cyclic AMP. The synthetic enzyme, adenyl cyclase is mainly associated with a particulate fraction which sediments at 7,000–30,000xg whereas the degradative enzyme, 3′5′ nucleotide phosphodiesterase, is soluble (does not sediment at 78,000xg). The adenyl cyclase activity is stimulated somewhat by prostaglandins and by catecholamines, agents which markedly stimulate cyclase in appropriate mammalian tissues. There is no detectable activity of guanyl cyclase, the enzyme which synthesizes cyclic GMP. $\text{Euglena}$ also contains a cyclic AMP stimulated protein kinase which is associated with a particulate fraction sedimenting at 30,000xg.     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface

The fluorescence properties of chlorophyll $\text{a}$ and $\text{b}$ monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorophyll molecules. Over the range of the mean distances from 27 to 21 Å, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll $\text{a}$ were observed. In the case of chlorophyll $\text{b}$, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 Å. When the mean distance was 18 Å, the short wavelength component of chlorophyll $\text{b}$ disappeared, and only the long wavelength component was observed.     

In vitro formation of assimilatory nitrate reductase: presence of the constitutive component in bacteria

Cell-free extracts of a selected group of bacteria which are capable of metabolyzing dinitrogen and/or nitrate contain a soluble form of the constitutive component which is active in the $\text{in}\text{vitro}$ formation of NADPH-nitrate reductase when mixed with extracts of $\text{N.}\text{crassa}\text{,}\text{nit}\text{-}\text{1}$. The constitutive component in these extracts is dialyzable and is insensitive to trypsin and protease. The constitutive component which substitutes for the absence of the $\text{nit}\text{-}\text{1}\text{gene product in the}\text{in}\text{vitro}$ formation of NADPH-nitrate reductase is postulated to be a low molecular weight cofactor or polypeptide and is shown to be present in a number of unrelated bacteria.     

Proteolytic inactivation of a pentafunctional enzyme conjugate: coordinate protection by the first substrate.

The $\text{arom}$ pentafunctional enzyme conjugate of $\text{Neurospora crassa}$ was exposed to trypsin, chymotrypsin, or a protease preparation from $\text{Neurospora}$ in the presence and absence of the first substrate, 3-deoxy-D-$\text{arabino}$-heptulosonate 7-phosphate. It was found that the first substrate coordinately protects all five activities from proteolytic inactivation, which indicates a conformational change induced by this compound. In addition, the data presented are consistent with the “domain” theory of conjugate structure. It is also argued that coordinate protection may be of physiological significance.     

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.     

Influence of protease inhibitors and energy metabolism on intracellular protein breakdown in starving Escherichia coli

It has been reported that certain inhibitors of serine proteases block intracellular protein breakdown in $\text{E. coli}$ subjected to nutritional deprivation. We show here that the protease inhibitors p-toluene sulfonyl fluoride and pentamidine isethionate inhibit protein breakdown in $\text{E. coli}$ deprived of glucose, but not in bacteria starved for inorganic phosphate or ammonia. Furthermore, we find that the protease inhibitors cause a drastic decline in cellular ATP levels when glucose is omitted from the incubation medium. It is concluded that these protease inhibitors influence protein breakdown by interfering with cellular energy production, rather than by interacting with a specific serine protease.     

Evidence against proton gradient formation being the cause of chlorophyll fluorescence quenching by N-methylphenazonium methosulfate

In strong illumination, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU)-poisoned chloroplasts exhibit a high yield of chlorophyll fluorescence while $\text{P-700}$ turnover, proton uptake, and phosphorylation are inhibited and a pH gradient is undetectable. When 10 μM $\text{N-}\text{methylphenazonium}$ methosulfate (PMS) is included, the fluorescence yield in light is substantially reduced, and when 100 μM ascorbate is also included, the yield is diminished approximately to the level in darkness. Only very slight increases in $\text{P-700}$ turnover and proton uptake (but no detectable pH gradient) accompany the fluorescence yield decline.When 10 μM PMS and 15 mM ascorbate are added to poisoned chloroplasts (the oxygen concentration being greatly reduced), $\text{P-700}$ turnover, proton uptake, the pH gradient and phosphorylation all reach high levels. In this case, the yield of chlorophyll fluorescence is low and is the same in both light and dark. Further addition of an uncoupler eliminates proton uptake, the pH gradient and phosphorylation but does not significantly elevate the fluorescence yield. From these observations we suggest that, in DCMU-poisoned chloroplasts, the fluorescence quenching with PMS occurs by a mechanism unrelated to the generation of a phosphorylation potential.With chloroplasts unpoisoned by DCMU, PMS quenches fluorescence and considerably stimulates proton uptake, the pH gradient and phosphorylation. However, in this case, PMS serves to restore net electron transport.     

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.     

Light-harvesting systems of brown algae and diatoms. Isolation and characterization of chlorophyll ac and chlorophyll afucoxanthin pigment-protein complexes

The present study examined the protein associations and energy transfer characteristics of chlorophyll c and fucoxanthin which are the major light-harvesting pigments in the brown and diatomaceous algae. It was demonstrated that sodium dodecyl sulfate (SDS)-solubilized photosynthetic membranes of these species when subjected to SDS polyacrylamide gel electrophoresis yielded three spectrally distinct pigment-protein complexes. The slowest migrating zone was identical to complex I, the SDS-altered form of the P-700 chlorophyll a-protein. The zone of intermediate mobility contained chlorophyll c and chlorophyll a in a molar ratio of 2 : 1, possessed no fucoxanthin, and showed efficient energy transfer from chlorophyll c to chlorophyll a. The fastest migrating pigment-protein zone contained fucoxanthin and chlorophyll a, possessed no chlorophyll c, and showed efficient energy transfer from fucoxanthin to chlorophyll a. It is demonstrated that the chlorophyll $\text{a}\text{c-}\text{protein}$ and the chlorophyll $\text{a}\text{fucoxanthin-protein}$ complexes are common to the brown algae and diatoms examined, and likely share similar roles in the photosynthetic units of these species.