Phosphorus compounds, proteins, nuclease and acid phosphatase activities in isolated spinach chloroplasts

This paper deals with attempts to elaborate a simple method of spinach chloroplast isolation ensuring a high proportion of intact chloroplasts. We obtained 3 preparations of isolated chloroplasts. Several preliminary analyses of the obtained chloroplast fraction were also performed. Phosphorus compounds, total protein and the enzyme activities of RNase, DNase and GPase were determined. We found: 0,36-0,59% of RNA, 0,19-0,24% of DNA, 2,1-2,9% of phospholipids and 26-28% of protein. RNase activity was very high.

A sulphate metabolizing centre in Euglena mitochondria

We have previously shown that a sulphate activating system is present on the outside of the inner mitochondrial membrane of Euglena gracilis Klebs. var. bacillaris Cori, but efforts to couple this system to ATP produced from oxidative phosphorylation were unsuccessful. In the present work we show that the concentration of Pi ordinarily used to support oxidative phosphorylation in these mitochondria (10 mM) inhibits sulphate activation completely; by reducing the concentration of Pi 10-fold, both processes proceeded normally. Sulphate activation under these conditions is inhibited nearly completely by the uncouplers of oxidative phosphorylation dinitrophenol (0.1 mM) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (0.2 microM). Sulphate reduction to form free cysteine, most of which appears outside the organelle, and in the cysteine of mitochondrial protein can be demonstrated in the same preparations, is membrane-bound and is inhibited by chloramphenicol (100 micrograms/ml), NaN3 (5 mM), KCN (100 microM); dinitrophenol (0.1 mM) or CCCP (0.2 microM). Digitonin fractionation of the mitochondria into mitoplasts, outer membranes and an intermembrane fraction show that reduction of 35SO4(2-) to form free cysteine and cysteine of protein is located on the mitoplasts; adenosine 5′-phosphosulphate sulphotransferase, the first enzyme of sulphate reduction, is found in the same location. Sulphate activation is highly enriched in the mitochondrial fraction of Euglena; the small amount found in the chloroplast fraction can be attributed to mitochondrial contamination. Thus, in Euglena, sulphate activation and reduction are contained in a sulphate metabolizing centre on the outside of the mitochondrial inner membrane; this centre appears to supply the mitochondrion and the rest of the cell with the products of sulphate activation as well as with reduced sulphur in the form of cysteine. Mitochondria from wild-type Euglena cells and from W10BSmL, a mutant lacking plastids completely, appear to be similar in the properties studied.

An investigation of photosynthetic sucrose production in bean leaves

First trifoliate leaves of wax beans. Phaseolus vulgaris, were exposed to 14CO2 in light for periods of 15 to 75 s. Leaves were frozen in liquid N2 and a chloroplast fraction was isolated nonaqueously. The chloroplast fraction contained a small amount of contaminating cytoplasm that could not be removed. Labelled photosynthetic compounds were separated from the whole leaves and from the corresponding chloroplast fractions and their radioactivity was measured.The compounds fell into two groups in their behaviour. Glyceric acid and ribulose, derived from photosynthetic 3-phosphoglyceric acid and ribulose diphosphate respectively, remained largely or exclusively in the chloroplast fraction. Labelled sucrose and serine and glycine, on the other hand, were located in the chloroplast fraction only during the first 15–30 s of 14CO2 supply, and appeared in the cytoplasmic fraction in increasing amounts thereafter. The results suggest that serine and glycine are produced in organelles in the layer of cytoplasm that closely surrounds the chloroplasts and is isolated with them in the non aqueous technique. Sucrose may also be synthesized from photosynthetic intermediates in this layer of cytoplasm. Sucrose was not formed from intermediates of the glycolate pathway in detached leaves.

Studies on the camparative physiology of Chara corallina. III. Nitrogen relations of internodal cell components during internodal cell expansion

Changes in the total nitrogen content of single internodal cells and in the nitrogen content of the cell wall, chloroplast fraction, TCA-insoluble cytoplasmic fraction, cytoplasmic sap, and vacuolar sap during the growth of the internodal cell are described. The nitrogen content of all fractions increases as the cells expand from c. 10 �l to over 200 �l in volume, and protein increases in the cytoplasm, the chloroplast fraction, and the cell wall. Cell wall nitrogen accounts for over half the total nitrogen of the cell and the bulk of the soluble nitrogen is present in the cytoplasm; only about 1% is found in the vacuole. The observations are discussed in relation to the cells of higher plants.

Studies on S-adenosylmethionine–magnesium protoporphyrin methyltransferase in Euglena gracilis strain Z

1. An enzyme that methylates magnesium protoporphyrin was detected in extracts of light-grown and dark-grown cells of Euglena gracilis. The activity in light-grown cells is two to three times that in cells grown in the dark. 2. The activity is mainly located in the chloroplast fraction from light-grown cells and in proplastids in dark-grown cells. However, in cells grown either in the light or dark, about 15–20% is found in particle-free supernatant. 3. The chloroplast methylating enzyme was solubilized by the action of Tween 80 and partially purified. The properties were investigated. 4. From experiments in which etiolated cells were illuminated in the presence of inhibitors of chloroplast or cytoplasmic protein synthesis, it appears that the methylating enzyme is made on cytoplasmic ribosomes.

The intracellular distribution of free nucleotides in the tobacco leaf. Formation of adenosine 5′-phosphate from adenosine 5′-triphosphate in the chloroplasts

1. A method of studying the free nucleotides of leaf tissue is outlined and the need for a metal ion-binding agent for the complete extraction of certain nucleotides by aqueous ethanol is established. 2. The method was used to study the effect of illumination or darkening of tobacco plants on the free nucleotides present in the chloroplast and non-chloroplast tissue components. 3. When plants that had been in the dark for a prolonged period were given 30sec. of bright light there was a rapid phosphorylation of ADP to ATP in both chloroplast and non-chloroplast components of the tissue. 4. Where plants were moved into the dark from conditions suitable for rapid photosynthesis there was a rapid conversion of ATP into AMP and the AMP was formed only in the chloroplast fraction. In continuing darkness the AMP remained restricted mainly to the chloroplast fraction for at least 2min., but eventually its concentration fell to the low value that is typical of tobacco leaves during conditions of constant illumination. If the plants were returned to the light for 30sec. the AMP was rapidly rephosphorylated.