Tracer and freeze-etching analysis of intra-cellular membrane-junctions in Paramecium

Abstract

In paramecia the membranes of alveoli and trichocysts are permanently connected to the cell membrane by membrane-junctions, which consist of membrane-intercalated particles in a regular geometrical arrangement. Trichocysts contain secretory material discharged by exocytosis. In unfixed or fixed cells these two compartments were impermeable to the following tracers: To "microperoxidases", i.e. a cytochrome c-derived heme-nonapeptide and a heme-undecapeptide (WM approximately 1650, 1900) applied in vivo, as well as to lanthanum and cytochrome c used during (La) or after (cytochrome c) fixation. The heme-nonapeptide was prepared by TPCK trypsin digestion of cytochrome c and subsequent purification by Sephadex gel chromatography--a simple and inexpensive new procedure resulting in preparations of high yield and purity. Tracers entered alveoli only when the plasmalemma and the alveolar membranes ruptured upon glutardialdehyde fixation. In no case were transmembraneous channels detectable in regions containing membrane-intercalated particles; this holds true for all tracers used and for freeze-fracture replicas obtained by tantalum-tungsten evaporation. With regard to attachment sites over trichocysts our results do not support the assumptions by others according to which exocytosis would be driven by an osmotic shift via transmembraneous channels (which would be analogous to inter-cellular coupling phenomena mediated by gap-junctions), unless such channels would be assumed to operate as carriers rather than via diffusion. Tracers did not penetrate trichocysts before exocytosis occurred. The functional role of membrane-intercalated particles on trichocyst attachments remains unclear. Despite some resemblance with gap-junctions all types of intra-cellular membrane-junctions investigated are functionally "tight" at the level of "resolution" obtained with tantalumtungsten-shadowing and with the tracers used.

Insights into porphyrin chemistry provided by the microperoxidases, the haempeptides derived from cytochrome c

“…43 Treatment with pepsin produces MP11 as virtually the only haem-containing peptide; trypsin produces MP9; and pepsin followed by trypsin produces MP8 with a small amount of MP6 as well. [22][23][24][44][45][46][47][48][49][50][51] The haempeptides are usually purified by gel permeation chromatography 24,52 or on a bovine serum albumin-Sepharose affinity column 53,54 with a final purification achieved by precipitation using ammonium sulfate near the isoelectric point of the particular haempeptide 46,52 (pI ≈ 4.5-5.0 22,23,55, 56 ).…”

Heme-Undecapeptide Labeling on Insulin for the Immunoassay of Insulin with Chemiluminescence Detection

“…12 When the reaction conditions optimal for the simultaneous introduction of the ATG on the Gly(A1) and Phe(B1)-amino groups of insulin with SATA were applied to a modification of compound 3, the ATG group was introduced into only the Phe(B1)-amino group to produce Gly(A1)-THPPhe(B1)-ATG-insulin (5). Gly(A1)Phe(B1)-(THP) 2 -insulin (4) was successfully used for the site-directed introduction of ATG on the Lys(B29)-amino group to produce Gly(A1)Phe(B1)-(THP) 2 -Lys(B29)-ATGinsulin (6). After removal of the THP-group from 5 and 6, compounds 8 and 9 were obtained by an 8-h treatment with 100 mM acetic acid, respectively.…”

Hydrophobic and Hydrophilic Radio-Iodination, Crosslinking, and Differential Extraction of Cell Surface Proteins in Paramecium tetraurelia Cells

“…Another aspect concerns preformed trichocyst exocytosis sites which, in Paramecium contain a group of 7 to 8 "rosette" particles [34] identified as integral proteins [50]. On high resolution Ta/W replicas, one such particle measures, with the metal pile-up subtracted, ∼12 nm [35]. Assuming a density of 1.2, a global molecular mass of ∼650 kDa would result [36].…”