Distribution of ATPases in wheat root membranes separated by phase partition

The distribution of divalent cation stimulated ATPase activity in relation to the distribution of other enzyme activities was studied for membrane fractions from wheat roots ( Tritium aestivum L . cv. Svenno). A homogenate from dark grown plants was fractionated by differential centrifugation at 1000 g , 10,000 g , 30,000 g and 60,000 g (1, 10, 30 and 60 KP fractions), followed by partition in an aqueous polymer two-phase system, using polyethylene glycol 4000/dextran T500 concentrations of 5.7/5.7, 5.9/5.9, 6.1/6.1, 6.3/6.3 and 6.5/6.5% (w/w). The 30 KP fraction was also separated by counter-current distribution id a 6.3/6.3% two-phase system. Protein and activities of Ca²⁺, Mg²⁺, and Mn²⁺ stimulated ATPases. cytochrome oxidase, light induced absorbance change (LIAC) related to cyt b reductions, inosine diphosphatase and NADH dependent antimycin A insensitive cytochrome c reductase were measured.
The partition of ATPase activities stimulated by Ca²⁺, Mg²⁺ or Mn²⁺ was similar at all polymer concentrations tested, indicating: a low cation specificity of the dominating ATPases. The distribution of ATPases. agreed with different marker enzymes in different centrifuge fractions. Divalent cation stimulated ATPases were evidently related to several of the organelles. In the different fractions the distribution of ATPase activity should then follow that of the marker enzyme of the dominant organelle. From studies with different polymer concentrations the 6.3/6.3-system was selected for further separation of the membranes in the 30 KP fraction by counter-current distribution. By this method one fraction was obtained, which probably consisted of plasmalemma and was free from mitochondrial material. Indications for plasmalemma in this fraction were a) similar partition as protoplasts and b) high LIAC activity.     

Binding of phytochrome to plasma membranes in vivo

A microsomal fraction was prepared by differential centrifugation from the homogenate of dark grown shoots of oats ( Avena sativa L. cv. Sol II). Plasma membranes were prepared from the microsomal fraction by means of an aqueous polymer two phase partition method. The content of phytochrome in the microsomal fraction and the plasma membrane fraction, respectively, were studied after different irradiation treatments of the intact shoots. Red irradiation increased the content of phytochrome in both the microsomal and plasma membrane fraction, especially in the presence of Mg²⁺. The increase induced by red light was fully reversible by far-red light for the plasma membrane fraction both in the presence and absence of Mg²⁺, in contrast to the microsomal fraction where Mg²⁺ had to be omitted. KI treatment of the membranes destroyed the binding of phytochrome whereas agents such as KCI, EDTA, CaCl₂ and Triton X-100, did not have this effect, indicating that the phytochrome attachment to the membrane is hydrophobic. This in vivo binding resembles to a large extent the one obtained in vitro by Sundqvist and Widell (Physiol. Plant. 59: 35–41, 1983) even though some differences between the phytochrome species and the membrane side exposed probably occur; so that the present interaction between phytochrome and the plasma membrane does not necessarily reflect the interaction that leads to physiological responses, and there could be more than one type of interaction.     

Fluorescence properties of plasma membranes from oats and cauliflower in relation to blue light physiology

Fluorescence properties of plasma membranes from dark-grown oat shoots ( Avena saliva L. cv. Sol II) and from cauliflower inflorescences ( Brassica oleracea L.) were investigated. Along with a flavin (with a possible connection to blue light physiology), a blue fluorescing component was present. The effect of NaN₃, phenyl acetic acid (PAA), KI (flavin inhibitors) and salicylhydroxamic acid (SHAM; inhibitor of e.g. the blue light-induced cytochrome b reduction) were followed with regard to the fluorescence properties of the two components as well as with regard to the light-induced cytochrome b reduction (LIAC). A change in flavin fluorescence and LIAC occurred at about the same concentration of PAA and SHAM, while LIAC was much more sensitive to KI and NaN₃ than was the fluorescence. Rapid freezing and thawing did not change the relative fluorescence emission from the flavin and blue fluorescing component, respectively, but storage at -20°C for one or two days increased the fluorescence, especially from the latter. There did not seem to be a tight coupling between the fluorescence properties of the blue fluorescing component (spectrally similar to a pteridine) and the flavin. Therefore, no conclusions could be drawn concerning their connection in blue light physiology, i.e. in processes such as phototropism.     

Separation of presumptive plasma membranes from mitochondria by partition in an aqueous polymer two-phase system

Light-induced absorbance changes (LIAC), indicating the reversible reduction of a b-type cytochrome, and with a possible connection to blue light photomorphogenesis, have been found in a presumptive plasma membrane rich centrifuge fraction from LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results technique membrane particles are separated according to differences in surface properties rather than size and density. LIAC could be separated into two fractions: one partitioning into the polyethylene glycol rich upper phase and another preferring the dextram rich lower phase. Mitochondria (cytochrome c oxidase) were recovered in the lower phase. A dual distribution of LIAC was found with all materials tested: corn coleoptiles, corn shoots, barley shoots and cauliflower inflorescences. About 80–90% of the cytochromes in the upper phase were related to LIAC, whereas only 10–15% of those in the lower phase were of this kind. The LIAC preferring the upper phase was probably bound to the plasma membrane, since plasma membrane vesicles are known to have a high partition in these phase systems. The lower phase LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results within one hour after the initial pelleting) for purification of presumptive plasma membranes, yielding a preparation which contained five times less mitochondrial contamination than the preparation obtained with sucrose gradient centrifugation (the 33/45% w/w sucrose interface fraction).     

Characterization of ferric reducing activity in roots of Fe-deficient Phaseolus vulgaris

Light-induced absorbance changes [LIAC; measured as Δ( A ₄₂₈– A ₄₁₀)] reflecting the reduction of a b -type cytochrome and mediated by an endogenous blue light absorbing receptor have been proposed to be related to blue light physiology of fungi and higher plants. It has also been suggested that the same cytochrome specifically can be reduced by red light in the presence of methylene blue. We have investigated the distribution of LIAC between different membrane fractions from corn ( Zea mays L.) coleoptiles and cauliflower ( Brassica oleracea L.) inflorescences. The membrane fractions were obtained by differential centrifugation followed by partition in an aqueous polymer two-phase system. By this procedure fractions rich in plasma membrane were obtained from both mitochondrial and microsomal fractions obtained by centrifugation. LIAC was by far most enriched in fractions also enriched in plasma membranes (identified by silicotungstic acid staining), but LIAC could be obtained also in other fractions. Our conclusion is that LIAC undoubtedly is caused by a b -cytochrome bound to the plasma membrane, but that LIAC also may be due to other b -cytochromes, one of which is probably located in the endoplasmic reticulum. Thus, the two assay procedures used for LIAC (blue and red light induced) could not disciminate between different b -cytochromes giving rise to LIAC.