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.     

Mechanism of gating of T-type calcium channels

We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.     

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.     

The photocycle and the structure of iron containing bacteriorhodopsin —a kinetic and Mössbauer spectroscopy investigation

Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe²⁺ or Fe³⁺ ions. ⁵⁷Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3⁺ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2⁺ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectroscopy show that the purple membrane becomes flexible only above 220 K. At the interface between membrane and bulk water the mobility is comparable to that of proteins with hydrophilic surfaces. The photocycle of Fe ³⁺-bR is slowed down compared to native bR. 3–5 Fe³⁺/bR are sufficient to inhibit the photocycle turnover by one order of magnitude. This specific effect is also found with Cr³⁺, though it is less pronounced. Mössbauer spectra of Fe³⁺-bR at 4 K reveal that iron nuclei are spin-coupled, indicating their close spatial proximity. It is proposed that iron trinuclear clusters interact with the proton uptake site of bR. Offprint requests to: M. Engelhard     

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.     

Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae)

Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA ethanolic phosphotungstic acid - PA periodic acid - UA/Pb uranyl acetate/lead