Chloroplast Phosphofructokinase: II. Partial Purification, Kinetic and Regulatory Properties

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP(2-) (Michaelis constant 30 mum). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism.     

Studies on a Maize Mutant Sensitive to Low Temperature II. Chloroplast Structure, Development, and Physiology

In the Zea mays L. mutant M11 grown in the dark at 15°, the ultrastructure of the etioplast is abnormal. The pigment content of the etioplasts is reduced but the in vivo absorption characteristics suggest that the normal protochlorophyll (ide)-holochrome is present. The lowered synthetic ability of the etioplasts is not primarily due to a reduced complement of plastid ribosomes. The plastids of mutant M11 grown in the light at 15° contain little pigment, are markedly deficient in ribosomes and their ultrastructure is abnormal. In mutant M11 grown at 15°, an extreme sensitivity of the plastid membranes to light was observed.     

Chloroplast inheritance and DNA variation in sweet, sour, and ground cherry

Sour cherry (Prunus cerasus L.) is an allotetraploid and both sweet cherry (P avium L.) and ground cherry (P. fruticosa Pall.) are the proposed progenitor species. The study investigated the maternal species origin(s) of sour cherry using chloroplast DNA (cpDNA) markers and a diverse set of 22 sweet, 25 sour, and 7 ground cherry selections. Two cpDNA restriction fragment length polymorphisms (RFLPs) and one polymerase chain reaction (PCR) fragment length polymorphism were identified among the 54 selections. The three polymorphisms considered together resolved four haplotypes. Analysis of sour cherry progeny indicated that the chloroplast genome is maternally inherited and therefore appropriate to use in determining maternal phylogenetic relationships. Ground cherry was found more likely than sweet cherry to be the maternal progenitor species of sour cherry since 23 of 25 of the sour cherry selections had the most prevalent ground cherry haplotype. However, the other two sour cherry selections tested had the most prevalent sweet cherry haplotype and a wild French sweet cherry selection had the most prevalent ground cherry haplotype. The results underscore the importance of using diverse Prunus germplasm to investigate phylogenetic relationships.     

Structure and Function of Chloroplast Membrane II. The Effects of Potassium and Magnesium Ions on the Absorption Spectrum and Photosystem II Function of Two Types of Chloroplast Me-mbranes

After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr., we obtained an incompletely developed chloroplast membrane. It was then compared with a completely developed chloroplast membrane obtaining from wheat seedlings grown under normal light-dark regime. We investgated the effect of various cations and their concentrations on the absorption spectrum and the photosystem Ⅱ function of the above two types of chloroplast membranes. A similar effect of potassium and magnesium ions on the absorption spectra of completely developed chloroplast membrane was observed. They decreased the absorption peak values at both the red and blue regions of the chloroplast membrane in the same manner. The degree of decrease in the peak value is proportional to ion concentration. But in the incompletely developed chloroplast membranes similar phenomenon was not observed. In the presence of K+ and Mg2+ of various concentrations, the absorptionn peaks at the red region overlapped almost completely, and these at the blue region only changed slightly with ion concentrations. DCIP photoreduction rate of the two types of chloroplast membranes was stimulated by the addition of K+ and Mg2+ in various concentrations. But the degree of stimulation in the two types of membranes was quite different. In the presence of l00 mM KCl or 5.0 mM MgCl2, DCIP photoreduction rate of completely developed chloroplast membranes was enhanced by 76.8% and 68.9% respectively, whereas in incompletely developed chloroplast membranes it was only increased by 56.3% and 36.4% respectively. The causes of the effects of cations on the absorption spectrum and the photosystem Ⅱ function of two types of chloroplast membranes were discussed.     

The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins

Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis.     

Structure and enzymology of Delta5-3-ketosteroid isomerase.

The three-dimensional structures of Delta5-3-ketosteroid isomerases from two different bacterial species have been determined. The structures reveal an unusually apolar active site, in which each of several competitive inhibitors of the enzyme are held by two hydrogen bonds with the general acids Tyr14 and Asp99, and by hydrophobic interactions. The hydrogen bond between the Tyr14 hydroxyl and the C3 oxyanion of a transition-state analog is a low-barrier hydrogen bond, as indicated by a highly deshielded nuclear magnetic resonance. Structural and other biochemical studies have enabled the proposal of a detailed catalytic mechanism for Delta5-3-ketosteroid isomerase and provided a major thrust towards understanding the mechanism not only in chemical terms but also in energetics terms.     

Photoinhibition of Chloroplast Reactions. II. Multiple Effects

Ultraviolet light inhibits the photoreduction of 2,6-dichlorophenolindo-phenol or nicotinamide adenine dinucleotide phosphate with water as the electron donor (evolution of oxygen) but not the photoreduction of nicotinamide adenine dinucleotide phosphate with ascorbate as the electron donor. It inhibits photophosphorylation associated with either system. Experiments undertaken to test whether plastoquinone is the site of UV inhibition yielded inconclusive results.

Visible light (> 420 mμ) causes the loss of all chloroplast activities, photosystem I being more sensitive than system II. The data suggests 2 modes of action for visible light. The one sensitized by system II results in damage resembling that of UV light. The other, sensitized by system I, results in the destruction of the reaction center of this system.

     

The Development of Chloroplast Structure During Leaf Ontogeny

Advances achieved during last fifteen years in the understanding the development of chloroplast ultrastructure during natural leaf ontogeny are summarized. Life span of a typical C3 mesophyll cell chloroplast is outlined and placed into the scheme of cyclic plastid interrelationships. Possible modifications of this development by stresses, environmental factors or experimental treatments are also shown.     

Azadideoxyadenosine: synthesis, enzymology, and anti-HIV activity

Synthesis of an azanucleoside, a new analogue of dideoxyadenosine, is described. This compound is only slowly deaminated by mammalian adenosine deaminase and it is a substrate for adenosine kinase. It exhibits in vitro anti-HIV activity.     

Plants under Climatic Stress: II. Low Temperature, High Light Effects on Chloroplast Ultrastructure

Mesophyll chloroplasts of the C₄-pathway grasses Sorghum and Paspalum and of the C₃-pathway legume soybean undergo ultrastructural changes under moderate light intensities (170 w·m⁻², 400-700 nanometers) at a tme when photosynthesis is much reduced by low temperature (10 C). The pattern of ultrastructural change was similar in these species, despite some differences in the initial sites of low temperature action on photosynthesis and differences in their mechanisms of CO₂ fixation. Starch grains in the chloroplasts rapidly reduce in size when chilling stress is applied. At or before the time starch grains completely disappear the membranes of the individual stromal thylakoids close together, reducing the intraspace between them while the chloroplast as a whole begins to swell. Extensive granal stacking appears to hold the thylakoids in position for some time, causing initial swelling to occur in the zone of the peripheral reticulum, when present. At more advanced stages of swelling the thylakoid system unravels while the thylakoid intraspaces dilate markedly. Initial thylakoid intraspace contraction is tentatively ascribed to an increase in the transmembrane hydrogen ion gradient causing movement of cations and undissociated organic acids from the thylakoid intraspace to the stroma. Chloroplast swelling may be caused by a hold-up of some osmotically active photosynthetic product in the chloroplast stroma. After granal unraveling and redilation of the thylakoid intraspaces, chloroplasts appear similar to those isolated in low salt hypotonic media. At the initial stages of stress-induced ultrastructural change, a marked gradient in degree of chloroplast swelling is seen within and between cells, being most pronounced near the surface of the leaf directly exposed to light.