Erwin Latzko

Light induced activation of fructose-1,6-bisphosphatase in isolated intact chloroplasts

Abstract Chloroplasts isolated from spinach leaves previously held in darkness contained no fructosebisphosphatase activity measured at pH 7.5 or 7.9, although high activity at pH 8.8 was observed. Following illumination of these chloroplasts for 7 min, enzyme activity at pH 7.9 was clearly detected, and after 24 min illumination was equal to 36% of the pH 8.8 activity; at this time activity at pH 7.5 also became apparent. The activity at pH 8.8 was not affected by illumination. Similar activation of fructosebisphosphatase in isolated pea chloroplasts was recorded.

Partial Purification and Properties of Soluble Ascorbate Peroxidases From Pea Leaves

One nonenzymic and two enzymic forms of ascorbate peroxidase were found in pea leaves, and designated A, B and C. Form A was due to a low molecular weight, heat-stable component, and could be separated from the enzymic forms by gel filtration. Forms B and C were soluble proteins with an apparent molecular weight of 57,000. These two forms could be separated by cation-exchange chromatography on CM-Sephadex C-50. This technique was incorporated into a procedure for their partial purification. Several properties of B and C were found to be similar: they were active over a wide pH range (5 to 8), they displayed very high affinities for H(2)O(2) (Km<5 μM), and Km values for ascorbate (6.5 mM and 2.9 mM, respectively) were comparable to physiological concentrations of this substrate. These properties are considered conducive to the proposed physiological role of ascorbate peroxidase, viz prevention of H(2)O(2) accumulation.

Intracellular localization of phosphorylases in spinach and pea leaves.

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.

An electrogenic uniport mediates light-dependent Ca2+ influx into intact spinach chloroplasts

Light‐dependent Ca2+ influx into intact spinach chloroplasts, measured with the metallochromic indicator arsenazo III, is stimulated by uncouplers (FCCP, CCCP, nigericin) and inhibited by ruthenium red. The data presented demonstrate that light‐dependent Ca2+ influx into chloroplasts is electrogenic and mediated by a uniport‐type carrier. The characteristics of the carrier system are similar to those of the Ca2+ uniport of mitochondria.

Enzyme Activities and Products of CO2 Fixation in Various Photosynthetic Organs of Wheat and Oat

Summary Enzyme activities associated with the Calvin cycle of photosynthesis and with C 4 metabolism, and rates and products of CO 2 fixation were examined with leaves, peduncles, glumes, lemmas, paleae and pericarps of oat and two varieties of wheat («Svenno» from Sweden and «Lerma Rojo» from Mexico). All tissues achieved net CO 2 fixation through the Calvin cycle. Some capacity for C 4 metabolism was detected in the first leaf and, more noticeably, in the tissues surrounding the grain; the latter were also capable of relativelyhigh CO 2 assimilation by phosphoenolpyruvate carboxylase in darkness. Ribulose 1,5-di-phosphate carboxylase activity was greater in leaves, but phosphoenolpyruvate carboxylase activity tended to predominate in the photosynthetic organs of the wheat ear and oat panicle. The wheat pericarp contained pyruvate, Pi dikinase and a relatively high phosphoenolpyruvate carboxylase activity. Glumes, lemmas, paleae and the pericarp seem adapted for catching CO 2 respired by the developing grain in addition to contributing to net CO 2 fixation.

Inhibition of spinach-leaf phosphofructokinase by 2-phosphoglycollate.

A considerable amount of the CO? fixed during photosynthesis in green plants is utilised for the synthesis of starch in chloroplasts. The possibility that the subsequent degradation of this starch might involve formation of triose phosphates which can be easily exported to the cytoplasm [ 1 J was recently strengthened by the detection, in this laboratory, of phosphofructokinase activity in chloroplasts [2] . Experiments described in this communication have confirmed that phosphoglycollate, a proposed intermediate of photorespiration, inhibits the activity of spinach-leaf phosphofructokinase. Strong inhibition of the chloroplast enzyme may significantly hinder the degradation of chloroplast starch during daylight hours.

Partial separation and interconversion of NADH- and NADPH-linked activities of purified glyceraldehyde 3-phosphate dehydrogenase from spinach chloroplasts.

Chloroplasts contain GAPDH* active with NAD(H) or NADP(H) in both directions of glycolysis [1,2], but efforts to completely separate the NAD(H) activity from the NADP(H) activity have so far not been successful [3 -5 ] . We have now partially separated GAPDH into a high molecular weight form (MW 240 000) and a low molecular weight form (79 000) using chromatography on a Bio-Gel column. The higher molecular weight enzyme was active mainly with NADH while the lower molecular weight form was active predominantly with NADPH. Gel filtration in the presence of DTT resulted in conversion of high molecular weight GAPDH to a low molecular weight form. Both forms of GAPDH were composed of 2 different subunits (MW 42 000 and 39 000). In addition, during all gel filtrations a small protein factor able to activate chloroplast GAPDH was observed. These results are considered in relation to the regulation of chloroplast GAPDH during light-dark transitions.

Visualization of antigenic proteins blotted onto nitrocellulose using the immuno-gold-staining (IGS) method.

A new and simple method for the detection of antigenic proteins blotted onto nitrocellulose was developed. After transfer of spinach stromal proteins and purified phosphoribulokinase immunolabeling was performed with phosphoribulokinase antiserum, followed by a) Protein A-labeled colloidal gold particles, and b) by horseradish peroxidase conjugated Protein A and substrate mixture. The Protein A-Gold method is at least twofold more sensitive than the Protein A-peroxidase procedure. Incubation of immunolabeled nitrocellulose replicas with 0.1 M glycine, pH 2.2, removes the antibody-Protein A-Gold complexes quantitatively without influencing the antigenicity of the immobilized proteins. The replicas can be re-used for immunostaining with other antisera. The versatile applicability of the immuno-gold-staining method suggests that it is a true alternative to the peroxidase assay.

[62] Fructose-bisphosphatase from spinach leaf chloroplast and cytoplasm

Publisher Summary Photosynthetic cells require fructose-bisphosphatase (D-fructose-l,6-bisphosphate l-phosphohydrolase) both in the chloroplast and in the cytoplasm. This chapter describes an assay method and the purification procedure for the enzyme fructose-bisphosphatase from spinach leaf chloroplast and cytoplasm. Fructose-bisphosphatase may be assayed either colorimetrically by estimating the released P i , or spectrophotometrically by coupling the production of fructose 6-phosphate to the reduction of NADP + using the enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. Purification of chloroplast fructose-bisphosphatase involves preparation of spinach ( Spinacia oleracea ) leaves, addition of solid ammonium sulfate, fractionation again by addition of solid ammonium sulfate, diethylaminoethyl (DEAE)-Sephadex A-50 chromatography, and Sephadex G-200 chromatography. The first four steps of the purification procedures are common to both enzymes until chromatography on DEAE-Sephadex A-50 that separates the chloroplast enzyme from the cytoplasmic enzyme; thereafter, the procedures differ. The properties of the leaf cytoplasmic enzyme are more or less comparable to those of mammalian fructose-bisphosphatases but the chloroplast enzyme is unique in being insensitive to adenosine monophosphate (AMP) and highly sensitive to the redox state of its environment.

Einfluß von Wachstumsbedingungen und Entwicklung auf δ13C-Werte in verschiedenen Organen und Inhaltsstoffen von Weizen, Hafer und Mais

Summary δ 13 C values of different parts of the wheat plant were found to range between -25 and -34 °against PDP standard. For oat plants the variation of δ 13 C values appeared to be smaller. Wheat when grown at higher temperatures (approx. 30 °C) or under saline conditions showed an increase up to 2 °for the isotope discrimination. The isotope discrimination also changed with growth phase: it increased up to the 4th–5th leaf, but then usually decreased in plant parts above the 5th leaf. With both wheat and maize leaves a correlation between δ 13 C values and the relative activities of ribulosebisphosphate carboxylase and phosphoenolpyruvate carboxylase was found. Among the constituents of oat plants and of oat, wheat and maize grains, organic acids and sugars had the less negative δ 13 C values while the values for proteins and lipids were more negative, probably due to an additional carbon discrimination during biosynthesis of these substances. The results indicate that variations of δ 13 C values caused by ecological conditions can be traced back to changes in carboxylase pattern and to formation of secondary metabolites.