A procedure for estimating the surface potential of charged or neutral membranes with 8-anilino-1-naphthalenesulphonate probe. Adequacy of the Gouy-Chapman model

Using the fluorescent anion 8-anilino-1-naphthalenesulphonate (ANS) for determining the membrane surface potential necessitates that the intrinsic affinity constant K_i for the ANS sites be known. Two methods are presented which do not rely on a determination of K_i at high ionic strength. They are respectively applied to neutral membranes (egg phosphatidylcholine liposomes) and highly charged natural ones (horse bean microsomes and liposomes from their phospholipids). The value of K_i appears to be insensitive to the level of occupancy of the sites, the KCl concentration and the pH in large ranges. Furthermore, the classical Gouy-Chapman model seems to describe correctly the whole set of data, provided apparent mean molecular areas larger than the published crystallographic ones are admitted.     

6(5)CARBOXYFLUORESCEIN AS A TRACER OF PHLOEM SAP TRANSLOCATION

6(5)carboxyfluorescein (6(5)CF), a polar fluorescein with an apparent pK of 6.3, was introduced, as a pH 6.3 solution, into the apoplast of lamina or petioles of mature soybean leaves. Freehand sections were prepared at various times and immediately observed with a fluorescence microscope. 6(5)CF-associated fluorescence appeared in all sink organs, from shoot apex to roots. It was strictly confined to the phloem regions, even after 4 days. Its transport into young leaves ceased at approximately the time they underwent sink-to-source transition. It was never transported between two leaflets of the same leaf. Its transport was interrupted by phloem destruction. All these transport characteristics were highly reproducible, and were paralleled by those of ¹⁴C transport after application of (¹⁴C)sucrose to leaf surfaces. In contrast with 6(5)CF, fluorescein was transported between mature leaves, and between leaflets of the same leaf. It was not restricted to phloem, and often appeared in the xylem region. These results indicate that 6(5)CF can be used to monitor phloem sap translocation in real time, in short- and long-term experiments.     

Effect of HCO - 3 concentration in the absorption solution on the energetic coupling of H+-cotransports in roots of Zea mays L.

The effect of HCO ₃ ^- on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO₂ partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H⁺ excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO ₃ ^- accumulation were slowed down, while ⁸⁶Rb⁺ uptake and K⁺ accumulation rates were increased by HCO ₃ ^- . These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H⁺ excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO₂/H₂CO₃/HCO ₃ ^- accelerated the diffusion of equivalent H⁺ inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.Abbreviations FW fresh weight - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - J_aa acetic acid influx - J_K ⁺ K⁺ influx - J_Pi orthophosphate influx - Mes 2-(N-morpholino)ethanesulfonic acid - pCO₂ CO₂ partial pressure - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.     

Ultrastructural study of the Sertoli cell and the limiting membrane in the seminiferous tubule of the adult cryptorchid rat

Cryptorchidism was simulated in 13-15-day-old rats by severing the gubernaculum testis and fixing the testis to the abdominal wall. Ultrastructural examination of the testis was made 100 days after birth when a number of modifications to the seminiferous tubules were noted. Germ cells were scanty, with only occasional spermatogonia and primary spermatocytes persisting. The nuclei of Sertoli cells were regular and oval or indented in shape. Their cytoplasm was characterized by a rich smooth endoplasmic reticulum, lipid inclusions and mitochondria with tubulo-vesicular cristae indicative of stero?dogenic activity. The decrease in the number of the germ cells induced a membrane rearrangement with numerous tight junctions and interdigitations between the Sertoli cells. Sertoli cell-specific junctional complexes were very extensive. The lamina propria of the seminiferous tubule appeared thickened and folded and the multilayered basal lamina had complex folds. After fixation with glutaraldehyde containing lanthanum, the latter substance was identified in the basal intercellular spaces of the seminiferous tubules indicating that the blood-testis barrier remains functional in the intra-abdominal testis.     

Potassium stimulation of corn root plasmalemma ATPase : I. Hydrolytic activity of native vesicles and purified enzyme

Potassium stimulation of the plasmalemma (Zea mays L. var Mona) was studied by using a constant ionic strength to prevent indirect stimulation by the electrostatic effect of K⁺ salts. The transmembrane electrochemical H⁺ gradient was eliminated by using gramicidin. In these conditions, K⁺ stimulation was attributable to a direct effect of the cation on plasmalemma proteins. We used both native vesicles isolated on a sucrose cushion, and solubilized and purified ATPase from phase-partitioned plasmalemma, according to the method of T. Nagao, W. Sasakawa, and T. Sugiyama ([1987] Plant Cell Physiol 28: 1181-1186). The purified enzyme had a high specific activity (15 micromoles per minute per milligram protein), but was only about 20% stimulated by K⁺. In both preparations, potassium (in the range around 1 millimolar) specifically decreased two-fold the vanadate inhibition constant, and increased the maximum rate of ATP hydrolysis. In plasmalemma vesicles, the Eadie-Scatchard graph of the K⁺-dependent ATPase activity as a function of K⁺ concentration was linear only at constant ionic strength. The purified ATPase preparation appeared as two closely spaced bands in the 100 kilodalton region with isoelectric point about 6.5. Nevertheless, this biochemical heterogeneity seems unlikely to be related to K⁺ stimulation, since K⁺ modified neither the pH optimum of the activity (pH 6.5) nor the monophasic kinetics of the vanadate inhibition, in both native plasmalemma and purified enzyme preparation.     

Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L.: increased transport in membranes isolated from sulphur-starved plants

The characteristics of sulphate uptake into right-side-out plasma-membrane vesicles isolated from roots of Brassica napus L., Metzger, cv. Drakkar, and purified by aqueous polymer two-phase partitioning, were investigated. Sulphate uptake into the vesicles was driven by an artificially imposed pH gradient (acid outside), and could be observed for 5–10 min before a plateau was reached and no further net uptake occurred. The uptake was partially inhibited in the presence of depolarizing agents and little uptake was observed in the absence of an imposed pH gradient. Uptake was strongly pH-dependent, being greatest at more acidic pH. After imposition of a pH gradient, the capacity for uptake decreased slowly (t1/2>10 min). The uptake had a high-affinity component which was strongly dependent on the external proton concentration (K _m=10μM at pH 5.0, 64 μM at pH 6.5). The K _m for protons varied from 0.4–1.9 μM as the sulphate concentration was reduced from 33 to 1 μM. A low-affinity component was observed which could be resolved at low temperatures (0 °C). Microsomal membranes that partitioned into the lower phase of the two-phase system gave no indication of high-affinity sulphate transport. Sulphate uptake into plasma-membrane vesicles isolated from sulphur-starved plant material was approximately twofold greater than that observed in those isolated from sulphate-fed plant material. Isolated vesicles therefore mirror the well-known in-vivo response of roots, indicating an increase in the number of transporters to be, at least in part, the underlying cause of derepression.     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.