Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

A fluorescence study of the binding of cytochrome C to mixed-phospholipid microvesicles : evidence for a preferred orientation of the bound protein.

Kinetic and equilibrium experiments are reported on the binding of the fluorescent probe 1,8-anilino-naphtalene sulfonate (ANS) to microvesicles of natural lecithin containing 10 per cent of an anionic phospholipip (90 : 10 mixtures). Kinetics discriminated between fast binding to the outer leaflet of the bilayer and apparently slow binding to the inner leaflet controlled by the diffusion of the probe across the bilayer. The equilibrium distribution of ANS between the two leaflets was not dependent on the nature of the anionic species and the spectral properties of bound ANS were identical in all cases investigated. A hyperbolic saturation was observed allowing to propose an affinity scale for the binding of ANS to mixtures of lecithin with phosphatidic acid, phosphatidylinositol, and cardiolipin. The effects on binding of ionic strength and sodium dodecylsulfate were also considered. The binding of horse heart ferricytochrome c to ANS-labelled microvesicles was studied quantitatively making use of the quenching of the probes fluorescence by the heme. Perrin-F?rster energy transfer could be analysed on the basis of a simple model of the physical arrangement of the system which was elaborated from published data referring to ANS and cytochrome c binding to phospholipids. Experimental and theoretical computed values of the quenching efficiency were compared and led to conclude in favor of a preferred orientation of the heme crevice fully accessible from the external space at the lipid interface.     

Block by phencyclidine of acetylcholine and barium induced adrenal catecholamine secretion

Activation of the sympathetic system by phencyclidine (PCP) should result in catecholamine release from the adrenals. However, adrenalectomy does not reduce PCP-induced hypertension. In an attempt to rectify this inconsistency, the direct effects of PCP on the bovine adrenal medulla were examined. At (3×10^?6M), PCP reduced the acetylcholine-(ACh)-induced catecholamine release by 50%. Surprisingly, barium-induced secretion of catecholamines was also reduced by PCP. ACh-induced catecholamine release was not altered by 10^?3M 4-aminopyridine (4 AP), the potassium channel blocker. Thus, calcium antagonist actions of PCP and consequent block of catecholamine secretion from adrenal medulla may explain the lack of effect of adrenalectomy on PCP-induced hypertension. Possible contributions of calcium and/or potassium channel blockade to other manifestations of PCP overdosage are discussed.     

Transport properties of single-file pores with two conformational states.

Complex facilitative membrane transporters of specific ligands may operate via inner channels subject to conformational transitions. To describe some properties of these systems, we introduce here a kinetic model of coupled transport of two species, L and w, through a two-conformational pore. The basic assumptions of the model are: a) single-file of, at most, n molecules inside the channel; b) each pore state is open to one of the compartments only; c) there is at most only one vacancy per pore; d) inside the channel, a molecule of L occupies the same positions as a molecule of w; and e) there is at most only one molecule of L per pore. We develop a general representation of the kinetic diagram of the model that is formally similar to the one used to describe one-vacancy transport through a one-conformational single-file pore. In many cases of biological importance, L could be a hydrophilic (ionic or nonionic) ligand and w could be water. The model also finds application to describe solute (w) transport under saturation conditions. In this latter case, L would be another solute, or a tracer of w. We derive steady-state expressions for the fluxes of L and w, and for the permeability coefficients. The main results obtained from the analysis of the model are the following. 1) Under the condition of equilibrium of w, the expression derived for the flux of L is formally indistinguishable from the one obtainable from a standard four-state model of ligand transport mediated by a two-conformational transporter. 2) When L is a tracer of w, we can derive an expression for the ratio between the main isotope and tracer permeability coefficients (Pw/Pd). We find that the near-equilibrium permeability ratio satisfies (n - 1) < or = (Pw/Pd)eq < or = n, a result previously derived for the one-conformational, single-file pore for the case that n > or = 2. 3) The kinetic model studied here represents a generalization of the carrier concept. In fact, for the case that n = 1 (corresponding to the classical single-occupancy carrier), the near-equilibrium permeability ratio satisfies 0 < or = (Pw/Pd)eq < or = 1, which is characteristic of a carrier performing exchange-diffusion.