An antibiotic activity was extracted from the cell mass of the myxobacterium, Stiginatella aurantiaca strain Sg a15. The antibiotic was toxic for yeasts and filamentous fungi, but not for most bacteria. The compound had the molecular formula C30H4207, appears to be a new antibiotic, and was named stigmatellin. In addition to stigmatellin, the strain produced relatively large quantities of a second, structurally unrelated antibiotic, a mixture of three myxalamid homologues.During a screening of gliding bacteria for new antibiotics, the myxobacterium Stigmatella aurantiaca strain Sg a15 was found to produce activity against yeasts, filamentous fungi and several Gram-positive bacteria. Part of the activity appeared in the culture supernatant, and part within the cell mass. By thin-layer chromatography it could be shown, that the antibiotic activity was due to at least two different substances. One of the activities was identified as a mixture of three myxalamids, compounds which were recently isolated in our laboratory from the myxobacterium, Myxococcus xanthus strain Mx x121.2). The second antibiotic had a completely different chemical structure, proved to be new, and was named stigmatellin. In some fermentation experiments traces (up to 2%) of a stereoisomer of stigmatellin were also isolated. As it is not yet unequivocally proven whether this stereoisomer is a natural compound, we assume that we are dealing with one antibiotic only. In this paper we wish to report on the production and isolation of the different antibiotics of Sg a15, and on some physico-chemical and biological properties of stigmatellin. The structure elucidation of stigmatellin3) and its mode of action" will be published elsewhere.Organism Stigmatella aurantiaca strain Sg a15 (Dawid Stamm 1) was isolated in 1977 by Dr. W. DAWID, Bonn, from rotting wood collected in the Siebengebirge mountains near Bonn, FRG. Stock cultures (cell suspensions in peptone liquid medium) were stored in a deep freezer at 80°C or in liquid nitrogen. The organism was grown in standard peptone liquid medium (1 % peptone from casein, tryptically digested, from Merck, Darmstadt; 0.1 % MgSO4• 7H2O; pH 6.8). Batch cultures of 100 ml or of 500 ml in 250-m1 or 1,000-ml Erlenmeyers flasks, respectively, were incubated at 30°C on a rotary shaker at 160 rpm for 3-5 days. Addition of 0.05-0.2% yeast extract to the medium enhanced growth up to the four-fold cell mass yield. This stimulation proved to be due to vitamin B10 and thiamine. Strain Sg al5 was able to use various sugars, polysaccharides and acids of the citrate cycle as substrates. In a basal medium composed of Casamino Acids (Difco) 0.1 %, MgSO4 -71-120 0.1 %, (NH,)2S0, 0.1 %, CaCl2.2H2O 0.05 %,

We have measured Ca2+ uptake and Ca2+ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca2+ uptake into cells was monitored with a Ca2+ electrode, whereas Ca2+ uptake into membrane vesicles was measured with 45Ca2+. Using inhibitors of known action, such as the H+ ATPase inhibitors NBD-Cl and NEM, the Ca2+ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP3) and its analog inositol 1,4,5-trisphosphorothioate (IPS3), we could functionally differentiate two nonmitochondrial Ca2+ pools. Ca2+ uptake into the IP3-sensitive Ca2+ pool (IsCaP) occurs by a MgATP-dependent Ca2+ uptake mechanism that exchanges Ca2+ for H+ ions. In the absence of ATP Ca2+ uptake can occur to some extent at the expense of an H+ gradient that is established by a vacuolar-type MgATP-dependent H+ pump present in the same organelle. The other Ca2+ pool takes up Ca2+ by a vanadate-sensitive Ca2+ ATPase and is insensitive to IP3 (IisCaP). The IsCaP is filled at "higher" Ca2+ concentrations (approximately 10(-6) mol/liter) which may occur during stimulation. The low steady-state [Ca2+] of approximately 10(-7) mol/liter is adjusted by the IisCaP. It is speculated that both Ca2+ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.

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Using the method of dehydration and rehydration, rough endoplasmic reticulum (RER) vesicles, isolated by differential centrifugation, can be enlarged to giant liposomes with diameters ranging from 5 to 200 micron. Patch-clamp studies on these giant RER liposomes revealed the existence of a channel with a mean conductance of 260 +/- 7 pS (n = 23; 140 mmol/liter KCl on both sides). The channel is about four times more permeable for Cl- than for K+. Its activity is strongly voltage regulated. At low potentials (+/- 20 mV) the channel is predominantly in its open state with an open probability near 1.0, whereas it closes permanently at high positive and negative voltages (+/- 70 mV). The channel activity is not influenced by changing the free Ca2+ concentration from 1 mmol/liter to less than 10(-9) mol/liter on either side, and is also not affected by typical Cl- -channel blockers like diphenylamine-2-carboxylate (DPC, 1 mmol/liter) or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 1 mmol/liter). Another chloride channel with a single-channel conductance of 79 +/- 6 pS (n = 4) was less frequently observed. In the potential range of -80 to +40 mV this channel displayed no voltage-dependent gating. We assume that these anion channels are involved in the maintenance of electroneutrality during Ca2+ uptake in the RER.

In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (Km for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulation was abolished by protonophores, such as nigericin (10(-6) mol/liter) or CCCP (10(-5) mol/liter), and by inhibitors of nonmitochondrial H+ ATPases, such as NEM or NBD-Cl, at a concentration of 10(-5) mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10(-5) mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2 x 10(-3) mol/liter), a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10(-4) mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependent in the sequence Cl- greater than Br- greater than gluconate-; in the presence of SO2-4, CH3COO- or No-3, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+ greater than Li+ greater than Na+ = choline+. As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl- and a K+ conductance, as well as a small H+ conductance, were found in the microsomal membranes. When membranes containing the H+ pump were further purified by Percoll gradient centrifugation (ninefold enrichment compared to homogenate), no correlation with markers for endoplasmic reticulum, mitochondria, plasma membranes, zymogen granules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar- or "V" -type H+ ATPase and has most similarities to that described in endoplasmic reticulum, Golgi apparatus or endosomes.