Karlheinz Kreuzberg

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations.

Chloroplasts with high rates of photosynthetic O2 evolution (up to 120 μmol O2· (mg Chl)-1·h-1 compared with 130 μmol O2· (mg Chl)-1·h-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO2 concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O2 uptake could be observed in the dark by high- and low-CO2 adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 μmol photons·m-2·s-1 for high- and low-CO2 adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 μM for low-CO2 adapted chloroplasts, whereas high-CO2 adapted chloroplasts were not saturated even at 700 μM DIC. The concentrations of DIC required to reach half-saturated rates of net O2 evolution (Km(DIC)) was 31.1 and 156 μM DIC for low- and high-CO2 adapted chloroplasts, respectively. These results demonstrate that the CO2 concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.

COMPARTMENTED METABOLITE POOLS IN PROTOPLASTS FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII : CHANGES AFTER TRANSITION FROM AEROBIOSIS TO ANAEROBIOSIS IN THE DARK

A rapid fractionation method for determination of metabolite levels in the chloroplast and the extrachloroplast compartment of Chlamydomonas reinhardtii has been developed. Protoplasts containing one large chloroplast were fractionated by passing them through a multilayer gradient containing digitonin, polyacrylamide, and a mixture of silicone oil and bromodecane. Lysis of the plasma membrane and the separation of the chloroplasts from most of the extrachloroplast material was achieved within less than 5 s. The chloroplast enriched fraction was contaminated with 3% fumarase (mitochondria) and 13% phosphoenol pyruvate carboxylase (cytosol). Metabolites of the upper glycolytic chain were detected mainly in the chloroplasts, whereas 2-phosphoglycerate was found only in the extrachloroplast compartment. Analysis of changes in metabolite concentrations after transition to anaerobic conditions in the dark pointed to a regulation of carbohydrate catabolism by chloroplast phosphofructokinase and by cytosolic pyruvatekinase.

Kinetic properties of asn-glycerol-3-phosphate dehydrogenase purified from the unicellular alga Chlamydomonas reinhardtii

Abstract A sn -glycerol-3-phosphate dehydrogenase ( sn -glycerol-3-phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) has been purified from the unicellular green alga Chlamydomonas reinhardtii 3400-fold to a specific activity of 34 μmol/mg protein per min by a simple procedure involving two chromatographic steps on affinity dyes. The pH optimum for reduction of dihydroxyacetone phosphate was 6.8 and for glycerol 3-phosphate oxidation it was 9.5. In the direction of dihydroxyacetone phosphate reduction, the enzyme showed Michaelis-Menten kinetics. The enzyme reacted specifically with NADH and dihydroxyacetone phosphate as substrates with affinity constants of 16 and 12 μM, respectively. Product inhibition as well as competitive inhibition pattern indicated a random-bi-bi reaction mechanism for sn -glycerol-3-phosphate dehydrogenase from C. reinhardtii . The effective control of dihydroxyacetone reduction catalysed via this enzyme by ATP, P 1 and NAD gave evidence for a physiological role of the enzyme in plastidic glycolysis.

Oscillatory starch degradation and fermentation in the green alga Chlamydomonas reinhardii

Abstract After transition to anaerobiosis an oscillatory fermentation of plastidic starch is found in the green alga Chlamydomonas reinhardii . Oscillations are investigated by analysis of fluctuations in starch, fermentation products and metabolites. Period length, amplitudes and phase relationships are determined by a biometrical processing of the biochemical signals, which allows statistical treatment even under limitation of data points. The mean period length is 59 min and oscillations are detectable within 3 h after the onset of anaerobiosis. Metabolized starch carbon is found by 90% in the accumulating fermentation products as formate, acetate and ethanol. From the intermediates only pyruvate increases during fermentative oscillations. A phase shift of 180° between ATP and both, AMP and ADP, indicates activity of adenylate kinase and values for energy charge fluctuates between 0.70 and 0.89. Phase relations of glucose-6- P to both AMP and pyruvate are 190° and 56°, respectively, whereas that of pyruvate to ATP is 134°. Glucose-6- P concentration does not follow the rate of starch degradation. Phase relations of fermentation products to pyruvate indicates enzyme activities of formate pathway. High period length as observed during algal fermentation is thought to be generated by a complex rate control in a different compartmentalized glycolytic pathway.

Pyruvate degradation via pyruvate formate-lyase (EC 2.3.1.54) and the enzymes of formate fermentation in the green alga Chlorogonium elongatum

Formate was formed in extracts of Chlorogonium elongatum via direct cleavage of pyruvate by a pyruvate formate-lyase (PFL, EC 2.3.1.54). The conversion of PFL to the catalytically active form required S-adenosylmethionine, ferric (2+), photoreduced deazariboflavin as reductant, pyruvate as allosteric effector and strict anaerobic conditions. At the optimum pH (pH 8.0), PFL catalyzed formate formation, pyruvate synthesis and the isotope exchange from [14C]formate into pyruvate with rates of 30.0, 1.5 and 1.2 nmol min-1 mg-1 protein, respectively. Treatment of the active enzyme with O2 irreversibly inactivated PFL activity (half-time 2 min). In addition to PFL, the activities of phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), aldehyde dehydrogenase (CoA acetylating, EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) were also detected in extracts of C. elongatum. The occurrence of these enzymes indicates pyruvate degradation via a formate-fermentation pathway during anaerobiosis of algal cells in the dark.

Partial purification and properties of alcohol dehydrogenase from the unicellular green alga Chlamydomonas moewusii

Abstract Alcohol dehydrogenase from the unicellular green alga Chlamydomonas moewusii was purified ca 142-fold. Optimum enzyme activity was at pH 6.8 for NADH oxidation and at pH 9.1 for NAD reduction. The MW of the native enzyme was determined by gel filtration to be ca 94 000, whereas SDS gel electrophoresis indicated the existence of two enzyme subunits with a MW of 42 000. ADH activity was specific for NAD. The oxidation rate of different alcohols decreased with higher chain length compared to ethanol. Free sulphydryl groups as well as a metal ion were necessary for enzyme activity as seen by the influence of iodoacetamide and 1,10-phenanthroline, respectively. The Km values estimated for the basic substrates were NADH 1.1 x 10−4 M, acetaldehyde 1.25 x 10−3 M (both at pH 6.8) and NAD 1.25 x 10−4 M, ethanol 2.22 x 10−2 M at pH 8.9. Kinetic analysis indicated that a Theorell-Chance mechanism was involved. No isoenzymic forms were observed in autotrophic algal cells. The role of the enzyme in starch fermentation of C. moewusii is discussed.

Amplitude and period length of yeast NADH oscillations fermenting on different sugars in dependence of growth phase, starvation and hexose concentration

Abstract In yeast cells the dependence of amplitude and period of NADH oscillations on growth phase, starvation time and concentration of hexose fermented (D‐glucose of D‐fructose) has been studied. With both sugars sustained NADH oscillations could be induced during the first logarithmic growth phase only within a small range of starvation time (0.5 to 1.7 h). After transition to diauxic growth on ethanol, oscillations were obtained with D‐glucose from 0.5 to 6 h starvation time, whereas D‐fructose could induce oscillations only after 2 h of starvation. With D‐fructose instead of D‐glucose differences in period lengths of NADH oscillations resulted. These differences after D‐fructose feeding were always consistent and did not depend on the growth phase of the yeast culture. Amplitude and period lengths generally depended on starvation, resulting in stable period lengths after 3 h starvation whereas amplitudes stabilized after 5.3 h. With 0 to 50 mM hexose typical saturation kinetics resulted in amplitude...

Statistical analysis of NADH oscillations in the yeast saccharomyces carlsbergensis fermenting on different sugars

Abstract In order to detect hidden periodicities in glycolytic oscillations and to estimate period lengths the interactive program system TIMESDIA was applied. This program system contains a collection of mathematical procedures apt to the above purpose. The period length of glycolytic oscillations in a suspension of yeast cells depends on the kind of sugar which is fermented by the cells: with each of the four sugars D‐gluctose, D‐fructose, D‐mannose and sucrose different period lengths are obtained. These oscillations are nearly sinusoidal, since in a fourier expansion of the signals the first term exceeds the following ones by at least one order of magnitude. If mixtures of D‐glucose and D‐fructose are added to a cell suspension, the resulting period lengths are between the values which are characteristic for both sugars alone. These oscillations are monotonic too, they are not a superposition of two oscillations different in period length and amplitude, as was shown by the application of our program s...

Purification and properties of phosphotransacetylase from the eucaryotic green alga Chlorogonium elongatum

Abstract Phosphotransacetylase (EC 2.3.1.8) was detected in cell-free crude extracts of starch-fermenting eucaryotic green algae. The enzyme was purified from autotrophically grown Chlorogonium elongatum. The purified enzyme fraction, after affinity chromatography, shows a single protein band upon acrylamide gel electrophoresis and has a molecular weight of 280 000. It consists of six subunits of identical molecular weight (44 000). The pH and temperature optima for the eucaryotic phosphotransacetylase are 7.6 and 28°C, respectively. The Km values at 25°C (pH 7.6) for acetyl-CoA and phosphate are 0.078 mM and 5.440 mM, respectively, and in the reverse reaction (acetyl-CoA synthesis) for CoA and acetyl phosphate 0.093 mM and 0.310 mM, respectively. The maximum velocity of the forward reaction was 1627 nkat/mg protein and of the reverse reaction 8582 nkat/mg protein. The activity of the eucaryotic phosphotransacetylase strictly depends on the presence of univalent cations (ammonium, Ka = 9 mM; potassium, Ka = 12.5 mM). Inactivation studies with iodoacetamide and iodoacetic acid revealed the presence of an essential sulphhydryl group at the catalytic site. Arsenolytic and product inhibition studies indicate a rapid equilibrium random bi-bi reaction mechanism for the enzyme from C. elongatum. The control of the enzyme activity in the forward reaction by both pyruvate and NADH gives evidence for a physiological function of phosphotransacetylase in anaerobic energy metabolism of eucaryotic green algae rather than in aerobic acetate activation.

Sn-glycerol-3-phosphate is a product of starch degradation in isolated chloroplasts from Chlamydomonas reinhardii

Abstract Sn-glycerol-3-phosphate Sn-glycerol-3-phosphate accumulates during starch catabolism by intact isolated chloroplasts from Chlamydomonas reinhardii in the dark. Further products detected mainly were 3-phospho-glyceric acid and triose phosphates with a ratio between 3-phosphoglyceric acid, sn-glycerol-3-phosphate and triose phosphates of 1.0:0.9:0.2. The production of sn-glycerol-3-phosphate by a plastidic glycerol-3-phosphate dehydrogenase provides a reasonable mechanism for regenerating reducing equivalents during continuous starch breakdown in the chloroplast of the alga C. reinhardii.