Purification and Synthesis under Anaerobic Conditions of Rice Arginine Decarboxylase

Arginine decarboxylase (ADC; EC 4.1.1.19 [EC] ) which catalyzes thesynthesis of putrescine, is involved in the responses of plantsto stress. The enzyme was purified 1,561-fold from rice coleoptilesby steps that included ammonium sulfate fractionation, gel filtration,ion-exchange chromatography and chromatofocusing. The purifiedenzyme had a pI of 5.3, a molecular mass of 176 kDa and appearedto be composed of three subunits of 63 kDa. A polyclonal antibodywas raised in a rabbit and the IgG fraction was purified fromserum. On Western blots the antibody recognized the ADC fromboth rice and E. coli. Immunoprecipitation with the ADC-specificantibodies allowed detection of radiolabelled ADC in extractsfrom aerobically and anaerobically grown rice seedlings thathad been supplied with a mixture of ¹⁴C-amino acids. This resultis discussed in relation to the role of ADC under anaerobicconditions. (Received April 28, 1994; Accepted September 27, 1994)     

Cytoskeletal elements in cotton seed hair developmentin vitro: Their possible regulatory role in cell wall organization

Summary The localization and orientation of cytoskeletal elements in developing cotton fibres were studied by the indirect immunofluorescence and the dry cleaving technique. Microtubules are transversely arranged to the cell axis, most probably in a flat helix, in the cortex of expanding fibres. Since the innermost deposited cellulose microfibrils always show primarily the same orientation it is postulated that the microtubules control the transverse deposition of the cellulose fibrils. Little further cell expansion takes place during secondary wall formation and the microfibril pattern corresponds to that of the cortical microtubules,e.g., in the steepness of their helicoidal turns. Microtubules with a length of 7–20 m were observed, probably they are longer. The importance of microtubule length on microfibril deposition is discussed. The density of microtubule packing is in the range of 8–14 m^-1 as in other comparable cell types. In contrast to the microtubules, actin filaments are most likely longitudinally oriented during different phases of fibre development. The dry cleaving technique reveals numerous coated pits in the plasma membrane which are not crossed by microtubules. They seem to be linked to the latter by filamentous structures.     

Polyamines and Anaerobic Elongation of Rice Coleoptile

The role of polyamines in the anaerobic elongation of rice (Oryzasativa L.) coleoptiles was studied. The reduced growth of ricecoleoptiles under anoxic conditions was accompanied by a massiveaccumulation of free putrescine. Putrescine was synthesizedfrom arginine in a reaction catalyzed by arginine decarboxylase(ADC). The anoxic titer of putrescine was closely correlatedwith elongation of coleoptiles. In experiments in which putrescineand inhibitors [-difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO)] of the synthesis of polyamines were exogenously supplied,we demonstrated an absolute requirement for putrescine, synthesizedby ADC, for anaerobic elongation of coleoptiles. The presenceof exogenous putrescine (alone or in combination with DFMA)increased the rate of anaerobic elongation of coleoptile by30–40%. (Received December 1, 1988; Accepted June 19, 1989)     

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: Cholesterylphosphoryldimethylethanolamine

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca²⁺. It has a K_i of about 30 m. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca²⁺-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.Abbreviation CPD cholesterylphosphoryldimethylethanolamine     

Accumulation and Interconversion of Amino Acids in Rice Roots under Anoxia

In excised rice roots, anaerobic degradation of proteins gaverise to an increase of free-amino acids. Anoxic accumulationof alanine, -aminobutyric acid and proline was the consequenceof the interconversion of glutamate, aspartate and amides. Theshift in the composition of the amino acid pool appears to becaused by changes in keto acid levels. The role of reactionsinvolved in amino acid interconversion and the physiologicalsignificance of these interconversions are considered and discussed. (Received November 25, 1988; Accepted June 9, 1988)     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Root and leaf attributes accounting for the performance of fast- and slow-growing grasses at different nutrient supply

Despite their difference in potential growth rate, the slow-growing Brachypodium pinnatum and the fast-growing Dactylis glomerata co-occur in many nutrient-poor calcareous grasslands. They are known to respond differently to increasing levels of N and P. An experiment was designed to measure which characteristics are affected by nutrient supply and contribute to the ecological performance of these species. Nutrient acquisition and root and shoot traits of these grasses were studied in a garden experiment with nine nutrient treatments in a factorial design of 3 N and 3 P levels each. D. glomerata was superior to B. pinnatum in nutrient acquisition and growth in all treatments. B. pinnatum was especially poor in P acquisition. Both species responded to increasing N supply and to a lesser extent to increasing P supply by decreasing their root length and increasing their leaf area per total plant weight. D. glomerata showed a higher plasticity. In most treatments, the root length ratio (RLR) and the leaf area ratio (LAR) were higher for D. glomerata. A factorization of these parameters into components expressing biomass allocation, form (root fineness or leaf thickness) and density (dry matter content) shows that the low density of the biomass of D. glomerata was the main cause for the higher RLR and LAR. The biomass allocation to the roots showed a considerable plasticity but did not differ between the species. B. pinnatum had the highest leaf weight ratio. Root fineness was highly plastic in D. glomerata, the difference with B. pinnatum being mainly due to the thick roots of D. glomerata at high nutrient supply. The leaf area/leaf fresh weight ratio did not show any plasticity and was slightly higher for B. pinnatum. It is concluded, that the low density of the biomass of D. glomerata is the pivotal trait responsible for its faster growth at all nutrient levels. It enables simultaneously a good nutrient acquisition capacity by the roots as well as a superior carbon acquisition by the leaves. The high biomass density of B. pinnatum will then result in a lower nutrient requirement due to a slower turnover, which in the long term is advantageous under nutrient-poor conditions.     

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H⁺-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10⁻² to 40.6 × 10⁻²Ωm². The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10⁻² Fm⁻² compared to 0.77 × 10⁻² Fm⁻² of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system. Received: 22 October 1996/Revised: 16 January 1997     

Colorimetric determination of microgram quantities of polylysine by trypan blue precipitation

A simple and sensitive method for the determination of polylysine in solution is described. Polylysine is quantitatively precipitated with trypan blue. The absorbance of unbound dye in the supernatant is inversely proportional to the concentration of this polyamino acid. The precipitation is identical for all sizes of polylysine of molecular weight 13,000 or higher, and is prevented by the addition of either polyanions or serum. The measurable range of polylysine hydrobromide is between 1 and 10 micrograms/ml, which is about 10-fold lower than that by the published methyl orange precipitation method.     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.