Testing the “methanochondrion concept”: are nucleotides transported across internal membranes in Methanobacterium thermoautotrophicum?

In order to test the Methanochondrion concept, uptake of adenine nucleotides in various membrane preparations of Methanobacterium thermoautotrophicum was studied. The uptake showed properties which are in general interpreted as indicative of a transport mechanism: (i) kinetics in the time range of minutes, (ii) temperature dependence, (iii) substrate specificity and (iv) failure to remove the substrate by extensive washing.However, nucleotide transport as an interpretation of this uptake can definitely be excluded. Not only an exchange mechanism of the mitochondrial type, but also a general exchange or an uniport mechanism was ruled out. In contrast, the nucleotide uptake was shown to be actually a tight and specific binding of ADP and ATP to binding sites at the interior side of the cell membrane. This was conclusively demonstrated in protoplasts obtained from M. thermoautotrophicum cells. In these protoplasts which do not contain internal membranes also nucleotide binding was observed, but only after disruption of the plasma membrane by osmotic lysis, which leads to the exposure of binding sites.     

Evidence for a branched respiratory system with two cytochrome oxidases in autotrophically grown Alcaligenes latus

The respiratory system of chemolithoautotrophically-grown Alcaligenes latus contains a, b, and c type cytochromes. Two cytochrome oxidases were identified by their carbon monoxide difference spectra and their differing sensitivities to cyanide and carbon monoxide. The oxidases were cytochrome o and an a-type cytochrome. Ubiquinone was present in A. latus membranes and could be reduced by H₂. The quinone analogue, 2-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), was a strong inhibitor of the H₂ oxidase reaction, but did not prevent the reduction of either ubiquinone or the cytochromes.Abbreviations HQNO 2-heptyl-4-hydroxy-quinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine     

Effect of aging on the rate of axonal transport of choline-phosphoglycerides

The anterograde axonal transport of choline-phosphoglycerides was studied in sciatic nerve motoneurons of adult (3-month-old) and aged (24-month-old) rats. After the spinal cord injection of [2-³H]glycerol, choline-phosphoglycerides; the major phospholipid class was transported along the nerve. The axonal transport rate was determined by plotting the distance covered by the front of transported radioactivity as a function of the time employed. In aged animals the rate of the choline-phosphoglyceride anterograde axonal transport was about 68% lower than that of adults; furthermore, the rate slowed down along the nerve in the proximal-distal direction. This alterated axonal transport mechanism might contribute to the degenerative processes observed in distal regions of peripheral nerve fibers of aged animals.     

Comparison of sequences from the malB regions of Salmonella typhimurium and Enterobacter aerogenes with Escherichia coli K12: A potential new regulatory site in the interoperonic region

Summary The malE and malK genes from Salmonella typhimurium, and the MalEFG operon and a portion of malK from Enterobacter aerogenes were cloned and sequenced. Plasmid-borne malE genes from both species and the malF and malG genes from E. aerogenes were expressed normally in Escherichia coli, and their products function in maltose transport. This shows that the malB products from the three species are interchangeable, at least in the combinations tested. The general genetic organization of the malB region is conserved. Potential binding sites and distances between them are highly conserved in the regulatory intervals. An unexpected conserved region was detected, which we call the U box, and which could be another target for a regulatory protein. This hypothesis is supported by the presence of the U box in the regulatory, region of the pulA-malX operon in Klebsiella pneumoniae. The intergenic region between malE and malF from S. typhimurium and E. aerogenes, contains inverted repeats similar to the palindromic units (PU or REP) found at the same location in E. coli. The predicted amino acid sequence of the encoded proteins showed 90% or more identity in every pairwise comparison of species.     

The role of Mg2+ in proton transport by the tonoplast pyrophosphatase in Riccia fluitans vacuoles

The Mg²⁺-dependent activity of the tonoplast pyrophosphatase (PPase) was investigated by measuring proton transport and by using the acridine orange technique on intact vacuoles of the aquatic liverwort Riccia fluitans L. In solutions with both Mg²⁺ and pyrophosphate present, a number of complexes are formed, which could all influence the enzymatic and hence the transport activity of the PPase. Therefore, the individual concentrations of these complexes were calculated and their contributions to proton transport across the tonoplast were tested. From these experiments we conclude that Mg²⁺ has three different roles: (i) Mg²⁺ stimulates transport activity of the PPase. (ii) Mg₂PP_i inhibits PPase-mediated H⁺ transport, (iii) MgPP_i* (= MgPP_i^2-+ MgHPP_i^-) is the substrate with an apparent K_1/2= 5–10 μM, with no discrimination between MgPP_i^2- and MgHPP_i^-.     

Low temperature delays development of photosystem II activity in winter rye leaves

The ability of leaves to acclimate photosynthetically to low temperature was examined during leaf development in winter rye plants ( Secale cereale L. cv. Puma) grown at 20°C or at 6°C. All leaves grown at 6°C exhibit increased chlorophyll (Chl) levels per leaf area, higher rates of uncoupled, light-saturated photosystem I (PSI) electron transport, and slower increases in photosystem II (PSII) electron transport capacity, when compared with 20°C leaves. The stoiehiometry of PSI and PSII was estimated for each leaf age class by quantifying Chl in elcctrophorctic separations of Chl-protein complexes. The ratio of PSII/PSI electron transport in 20°C leaves is highly correlated with the ratio of core Chl a -proteins associated with PSII (CPa) to those associated with PSI (CP1). In contrast, PSII/PSI electron transport in 6°C leaves is not as well correlated with CPa/CP1 and is related, in part, to the amount and organization of light-harvesting Chl a/b -proteins associated with PSII. CPa/CP1 increases slowly in 6°C leaves, although the ratio of CPa/CP1 in mature 20°C and 6°C leaves is not different. The results suggest that increased PSI activity at low temperature is not related to an increase in the relative proportion of PSI and may reflect, instead, a regulatory change. Photosynthetic acclimation to low environmental temperature involves increased PSI activity in mature leaves shifted to 6°C. In leaves grown entirely at 6°C, however, acclimation includes both increased PSI activity and modifications in the rate of accumlation of PSII and in the organization of LHCII.     

Sucrose transport into the phloem of Ricinus communis L. seedlings as measured by the analysis of sieve-tube sap

Careful cutting of the hypocotyl of Ricinus communis L. seedlings led to the exudation of pure sieve-tube sap for 2–3 h. This offered the possibility of testing the phloem-loading system qualitatively and quantitatively by incubating the cotyledons with different solutes of various concentrations to determine whether or not these solutes were loaded into the sieve tubes. The concentration which was achieved by loading and the time course could also be documented. This study concentrated on the loading of sucrose because it is the major naturally translocated sieve-tube compound. The sucrose concentration of sieve-tube sap was approx. 300 mM when the cotyledons were buried in the endosperm. When the cotyledons were excised from the endosperm and incubated in buffer, the sucrose concentration decreased gradually to 80–100 mM. This sucrose level was maintained for several hours by starch breakdown. Incubation of the excised cotyledons in sucrose caused the sucrose concentration in the sieve tubes to rise from 80 to 400 mM, depending on the sucrose concentration in the medium. Thus the sucrose concentration in the sieve tubes could be manipulated over a wide range. The transfer of labelled sucrose to the sieve-tube sap took 10 min; full isotope equilibration was finally reached after 2 h. An increase of K⁺ in the medium or in the sieve tubes did not change the sucrose concentration in the sievetube sap. Similarly the experimentally induced change of sucrose concentration in the sieve tubes did not affect the K⁺ concentration in the exudate. High concentrations of K⁺, however, strongly reduced the flow rate of exudation. Similar results were obtained with Na⁺ (data not shown). The minimum translocation speed in the sieve tubes in vivo was calculated from the growth increment of the seedling to be 1.03 m·h^-1, a value, which on average was also obtained for the exudation system with the endosperm attached. This comparison of the in-vivo rate of phloem transport and the exudation rate from cut hypocotyls indicates that sink control of phloem transport in the seedlings of that particular age was small, if there was any at all, and that the results from the experimental exudation system were probably not falsified by removal of the sink tissues.Abbreviations PTS 3-hydroxy-5,8, 10-pyrenetrisulfonate     

Uptake of Lucifer Yellow CH into plant-cell protoplasts: a quantitative assessment of fluid-phase endocytosis

The highly fluorescent dye Lucifer Yellow CH (LYCH), now in common use in microinjection studies, has been shown to enter the vacuole of a range of plant-cell protoplasts from the external medium. Uptake was quantified by lysing the protoplasts following incubation and determining the amount of LYCH incorporated by spectrofluorimetry. Uptake was biphasic with respect to both time and substrate concentration, enhanced at low pH and inhibited by low temperature and metabolic inhibitors. The kinetics of uptake showed several similarities with those reported for the fluid-phase endocytosis of LYCH in animal cells and yeast cells. A calculated membrane permeability coefficient for LYCH, based on the observed rates of uptake, was too high to be consistent with simple diffusion of the undissociated form of the molecule and inconsistent with the membrane-impermeant properties of the dye. The data are discussed in the light of the possibility of fluid-phase endocytosis versus active transmembrane transport.Abbreviations CCCP carbonyl cyanide M-chlorophenyl hydrazone - LYCH Lucifer Yellow CH     

Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient