The uptake of radioactively labeled hexoses and pentoses into the sorbitol-impermeable 3H20 space (the space surrounded by the inner envelope membrne) of spinach (Spinacia oleracea L.) chloroplasts has been studied using silicone layer filtering centrifugation. Of the compounds tested, D-xylose, D-mannose, L-arabinose, and D-glucose are transported most rapidly, followed by D-fructose and L-arabinose. The rate of L-glucose uptake is only about 5% of that of D-glucose.

Intact chloroplasts of spinach (Spinacia oleracea L.) evolve O2 in the light in a glycerate-dependent reaction at rates usually close to 10 μmolxmg(-1) chlorophyllxh(-1). Glycerate isfirst phosphorylated and the resulting phosphoglycerate reduced to the sugar level. Products of the reaction are the intermediates of the Calvin cycle and glycolate. The ratio of triosephosphates to phosphoglycerate is higher under low light or at a low pH than under high light or at a high pH. Chloroplasts contain activities of glycerate kinase which approximately correspond to observed glycerate reduction rates at light saturation. The main part of the glycerate kinase of leaf cells is localized in the chloroplasts, but considerable activity also resides outside these organelles. Glycerate can enter intact chloroplasts of spinach as the anion and the undissociated acid. It can thus mediate indirect proton transfer across the chloroplast envelope. In the presence of slowly permeating cations it is taken up mainly in an anion exchange reaction. Chloride and acetate anions permeate faster than the glycerate anion. The relation between glycerate reduction and photorespiration is discussed.

The amino acid permeability of the envelope of intact, functional spinach (Spinacia oleracea L.) chloroplasts was investigated by light scattering, volumetry and uptake of (14)C-labelled amino acids. The criterion for the functionally of the chloroplasts was their ability to reduce CO2, PGA and oxaloacetate in the light at high rates.Net uptake into the chloroplast interior of neutral amino acids such as alanine, glycine, serine, proline, threonine or valine occurred only at very low rates. The uptake was concentration dependent, indicating unspecific diffusion rather than carrier-mediated transport. The slowness of uptake is emphasized by the capability of neutral amino acids to provide osmotic support for intact chloroplasts during a considerable length of time. Back-exchange experiments also failed to indicate the existence of specific exchange carriers for the transport of neutral amino acids such as alanine or glycine through the envelope of intact chloroplasts. Dicarboxylic amino-acids are known to be taken up by the so-called dicarboxylate translocator. The same carrier was found to catalyze also the transfer of asparagine and glutamine.The data do not support current assumptions concerning fast carrier-mediated transport of neutral amino acids and their role in the transfer of carbon during photosynthesis.

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Several protein fractions containing endonuclease activity against gemma-irradiated DNA (gamma-endonuclease) were isolated from M. luteus. The crude extract was eluted on a phosphocellulose column and chromatographed on TEAE cellulose and subsequently on hydroxyapatite. Five peaks of gamma-endonuclease were obtained from each preparation. Repeated experiments showed comparable chromatographic behavior of the fractions. There was no detectable activity of U.V.-endonuclease in the fractions with gamma-endonuclease but a small contamination of endonuclease against unirradiated DNA and against DNA with apurinic sites. The gamma-endonuclease is stimulated by, but is not dependent on, magnesium. Several tests for endonuclease activity have been used: the analysis of strand breaks in calf-thymus DNA or in PM2 DNA, and the determination of end-groups formed by endonuclease, either 3'OH end-groups or phosphomonoester end groups. From the results obtained it can be assumed that the strand breaks induced by the gamma-endonuclease carry 3'OH and 5' phosphate end groups.

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A protein fraction from Micrococcus luteus with endonuclease activity against gamma-irradiated DNA was isolated and characterized. An additional activity on apurinic sites could not be separated, either by sucrose gradient sedimentation or by gel filtration through Sephadex G 100. From gel filtration, a molecular weight of about 25 000 was calculated for both endonuclease activities. The endonuclease activity against gamma-irradiated DNA was stimulated five-fold with 5 mM Mg++, whereas that against apurinic sites was less dependent on the Mg++ concentration. 100 mM KCl inhibited the gamma-ray endonuclease, but not the apurinic endonuclease activity. In gamma-irradiated RNA the protein recognized 1.65 endonuclease sensitive sites per radiation induced single-strand break, among which are 0.45 alkali labile lesions in the nucleotide strand. The affinity of the enzyme for the endonuclease sensitive site was evaluated resulting in a Km-value of 73 nM.

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