Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.
The synthesis of acetyl-CoA from C02, H2, and various C1 compounds was studied in vitro with extracts and with protein fractions of Methanobacterium thermoautotrophicum. Acetyl-CoA synthesis from C 0 2 and H2 by extracts required C 0 2 reduction to CH4 to proceed. Both processes were highly stimulated by formaldehyde which served as the carbon precursor of both CH4 and the CH3 group of acetate. Carbon monoxide in combination with formaldehyde dramatically stimulated the acetyl-CoA synthesis up to 1 %-fold. In t h s system, which did not require C 0 2 reduction to the formaldehyde and CO level, acetyl-CoA synthesis was no longer dependent on CH4 formation. The soluble (100000 x g supernatant) cell protein was resolved into a protein fraction [45-60% (NH4),S04-fraction] which catalyzed acetyl-CoA synthesis at a specific rate of 15 nmol . min-' . (equivalent of mg cell protein)-' (60 "C). This oxygen-sensitive enzyme reaction required dithioerythritol for activity and was strictly dependent on (a) coenzyme A, (b) CO, and (c) N5,N"-methylene tetrahydromethanopterin, N5-methyl tetrahydromethanopterin or formaldehyde plus tetrahydromethanopterin. The incorporation of formaldehyde is explained by the spontaneous formation of methylene tetrahydromethanopterin. The product of the reaction, acetyl-CoA, was quantitatively derived from CO (carboxyl of acetate) and a C1 derivative of tetrahydromethanopterin (methyl of acetate). The C1 derivative of tetrahydromethanopterin could not be replaced by a C1 derivative of tetrahydrofolate or by methyl-coenzyme M; ATP was not required. The active protein fraction contained CO dehydrogenase and at least one corrinoid protein. These results provide strong biochemical arguments for the proposed mechanism of autotrophic acetyl-CoA synthesis in Methanobacterium.Methanogenic bacteria are strict anaerobic archaebacteria which in general are able to obtain energy simply from the reduction of C 0 2 with H2 (C02 + 4 H2 --t CH4 + 2 H20).Most species grow autotrophically, i.e. in addition they use CO, as sole carbon compound for biosyntheses [I]. In Methanobacterium thermoautotrophicum, a thermophilic microorganism [2], C 0 2 assimilation does not proceed via the pentose phosphate cycle (Calvin cycle) [3 -81. Rather, a novel non-cyclic pathway for a de novo synthesis of acetyl-coenzyme A from 2 C 0 2 + 4 H2 was established termed the 'acetylCoA pathway' [8 -1 I]. This process was found to have many features in common with acetate synthesis from CO, in the acetogenic bacterium Clostridium thermoaceticum (for reviews see [12-141. C. thermoaceticum ferments hexoses to acetates: two acetate molecules are generated from two pyruvate molecules and one from (20,. The validity of this model was questioned when carbon monoxide was found to replace pyruvate as C 0 2 donor, and acetylCoA was shown to be the reaction product [16]. Studies by the laboratory of Thauer [17 -201 had indicated that the nickel enzyme carbon monoxide dehydrogenase is involved in this process. It is now postulated that CO dehy...
Methanobacterium thermoautotrophicum, a methane forming archaebacterium, grows autotrophically by synthesizing activated acetic acid from 2 CO2. It is demonstrated in vitro that the methyl group of acetate is derived from methenyl tetrahydromethanopterin, which is known to be a one‐carbon carrying coenzyme in CO2 reduction to methane. The direct acetate precursors are suggested to be methyl tetrahydromethanopterin (“activated methanol”) and “activated carbon monoxide”.
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