Glycolate Pathway in Algae

No glycolate oxidase activity could be detected by manometric, isotopic, or spectrophotometric techniques in cell extracts from 5 strains of algae grown in the light with CO(2). However, NADH:glyoxylate reductase, phosphoglycolate phosphatase and isocitrate dehydrogenase were detected in the cell extracts. The serine formed by Chlorella or Chlamydomonas after 12 seconds of photosynthetic (14)CO(2) fixation contained 70 to 80% of its (14)C in the carboxyl carbon. This distribution of label in serine was similar to that in phosphoglycerate from the same experiment. Thus, in algae serine is probably formed directly from phosphoglycerate. These results differ from those of higher plants which form uniformly labeled serine from glycolate in short time periods when phosphoglycerate is still carboxyl labeled.In glycolate formed by algae in 5 and 10 seconds of (14)CO(2) fixation, C(2) was at least twice as radioactive as C(1). A similar skewed labeling in C(2) and C(3) of 3-phosphoglycerate and serine suggests a common precursor for glycolate and 3-phosphoglycerate. Glycine formed by the algae, however, from the same experiments was uniformly labeled.Manganese deficient Chlorella incorporated only 2% of the total (14)CO(2) fixed in 10 minutes into glycolate, while in normal Chlorella 30% of the total (14)C was found in glycolate. Manganese deficient Chlorella also accumulated more (14)C in glycine and serine.Glycolate excretion by Chlorella was maximal in 10 mm bicarbonate and occurred only in the light, and was not influenced by the addition of glycolate. No time dependent uptake of significant amounts of either glycolate or phosphoglycolate was observed. When small amounts of glycolate-2-(14)C were fed to Chlorella or Scenedesmus, only 2 to 3% was metabolized after 30 to 60 minutes. The algae were not capable of significant glycolate metabolism as is the higher plant.The failure to detect glycolate oxidase, the low level glycolate-(14)C metabolism, and the formation of serine from phosphoglycerate rather than from glycolate are consistent with the concept of an incomplete glycolate pathway in algae.     

Purification, characterization, and immunological properties for two isoforms of glutathione reductase from eastern white pine needles

Glutathione reductase (EC 1.6.4.2) was purified from Eastern white pine (Pinus strobus L.) needles. The purification steps included affinity chromatography using 2′, 5′-ADP-Sepharose, FPLC-anion-exchange, FPLC-hydrophobic interaction, and FPLC-gel filtration. Separation of proteins by FPLC-anion-exchange resulted in the recovery of two distinct isoforms of glutathione reductase (GR_A and GR_B). Purified GR_A had a specific activity of 1.81 microkatals per milligram of protein and GR_B had a specific activity of 6.08 microkatals per milligram of protein. GR_A accounted for 17% of the total units of glutathione reductase recovered after anion-exchange separation and GR_B accounted for 83%. The native molecular mass for GR_A was 103 to 104 kilodaltons and for GR_B was 88 to 95 kilodaltons. Both isoforms of glutathione reductase were dimers composed of identical subunit molecular masses which were 53 to 54 kilodaltons for GR_A and 57 kilodaltons for GR_B. The pH optimum for GR_A was 7.25 to 7.75 and for GR_B was 7.25. At 25°C the K_m for GSSG was 15.3 and 39.8 micromolar for GR_A and GR_B, respectively. For NADPH, the K_m was 3.7 and 8.8 micromolar for GR_A and GR_B, respectively. Antibody produced from purified GR_B was reactive with both native and denatured GR_B, but was cross-reactive with only native GR_A.     

Quantal analysis of long-term posttetanic changes in minimal postsynaptic potentials of hippocampal slices in vitro

Minimal excitatory postsynaptic potentials (EPSP) were investigated in 13 neurons under single or double-pulse near-threshold microstimulation of the radial layer (Schaffer's collaterals) and stratum oriens in surviving hippocampal slices (area CA1) in guinea pigs. The amplitude of 23 EPSP (9 units; 12 pathways) rose after tetanization of Schaffer's collaterals over a 5–55 min period, taken as long-term potentiation (LTP). Statistical analysis conducted using four methods of quantal hypothesis based on a binomial approximation revealed an increase in mean quantal content (m) during LTP. The rise in quantal size was only statistically significant when using data obtained from a section of these methods (mainly for stretches of over 15 min following tetanization) and shows no correlation with intensity of LTP. The pronounced rise in m demonstrated using different methods matches data from experiments on intact animals and indicates a presynaptic location of the mechanisms underlying protracted persistence of residual tetanization lasting some tens of minutes.Institute for Brain Research, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Max-Planck Institute of Biophysical Chemistry, Göttingen, Germany. Zoological Institute, Jagiellonian University, Cracow, Poland. Translated from Neirofiziologiya, Vol. 22, No. 6, pp. 752–761, November–December, 1990.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.