Sucrose-based Medium Research Articles

Ultrastructural aspects ofZymomonas mobilis during the alcoholic fermentation of sucrose-based media

A flocculent, high ethanol-producing strain ofZymomonas mobilis subsp.mobilis was isolated from spontaneously-fermenting sugar-cane juice. Cells collected during the stationary growth phase of a culture in sucrose based medium, were examined in thin section by electron microscopy and showed elongated crystalloid cytoplasmic structures, resembling eukaryote peroxisomes, but devoid of an external membrane. These structures have not been previously described inZymomonas. The formation of vesicles from an external membrane as well as zones of lysis were also observed. General considerations on the unusual structures are discussed in this report as well as their possible relationship between ethanol, sugar concentration and growth phases of the culture.

Improved procedure for the transformation of the entomopathic microorganism Bacillus sphaericus

Previous studies of the mosquito larvicidal bacterium Bacillus sphaericus 1593 have shown that this organism can be transformed with plasmid DNA, although at extremely low freqencies. In this paper, we report the use of a sucrose-based regeneration medium following plasmid transformation of B. sphaericus 1593 protoplasts. This modification greatly improved both the transformation frequency and the regeneration rate of the transformants.

Isolation of a stable apical segment of the guinea pig sperm acrosome.

The large apical segments of guinea pig sperm acrosomes were mechanically separated from the spermatozoa and subsequently isolated by density gradient centrifugation. The isolated acrosomal caps were very stable and maintained their crescent morphology when suspended in sucrose-based medium buffered at pH 5.6, with or without the acrosin inhibitor p-aminobenzamidine (pAB). Examination under the electron microscope showed that the acrosomal caps were free of plasma membrane and were bound by an outer acrosomal membrane which was discontinuous. Enzymatic analysis after lysis of the caps indicated that acrosin and hyaluronidase were present with high specific activity, while only a trace amount of acid phosphatase activity and no arylsulphatase, phospholipase A2, or phospholipase C activities were present. Significant particulate acrosin activity, but only trace amounts of soluble acrosin activity, could be detected in the isolated acrosomal caps if assayed immediately after isolation in the absence of pAB. However, soluble acrosin activity of high specific activity was obtained after the acrosomal caps were extracted by 10% glycerol buffered at low pH (pH 3.0). The new procedures provide a means to isolate and purify guinea pig sperm apical acrosomal segments rapidly.

Proton translocation by cytochrome oxidase in (antimycin + myxothiazol)-treated rat liver mitochondria using ferrocyanide or hexammineruthenium as electron donor.

When O2 was injected into an anaerobic suspension of valinomycin-treated rat liver mitochondria inhibited with rotenone, antimycin, and myxothiazol, a small amount of O2 (0.23-0.33 ng-atom of O/mg of protein) was reduced extremely rapidly (within the 2 s time-resolution of the oxygen electrode). The subsequent steady-state rate of flow of electrons to oxygen was very low [less than 3 nequiv. X s-1 X (g of mitochondrial protein)-1]. In the presence of valinomycin there was a rapid ejection of protons synchronous with the rapid phase of O2 consumption corresponding to 0.38-0.61 nequiv. of H+ X (mg of mitochondrial protein)-1. When valinomycin was replaced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) there was a rapid alkalification of the medium corresponding to 0.20-0.42 nequiv. of H+ X (mg of mitochondrial protein)-1. When 2 mM-Fe(CN)6(4-) was present to re-reduce endogenous cytochrome c, O2 consumption was still biphasic but the second phase of O2 consumption was very much more rapid [600 nequiv. X s-1 X (g of protein)-1], and resulted in the virtually complete consumption of the O2 in the pulse within 4 s. With 60 microM-Ru(NH3)6(2+) as reductant, O2 consumption was even faster [1200 nequiv. X s-1 X (g of protein)-1]. In a medium containing 150 mM-choline chloride with Ru(NH3)6(2+) as reductant, the proton per reducing equivalent stoichiometry (delta H+O/e-) was +0.95 in the presence of valinomycin and -0.94 in the presence of FCCP. In choline chloride medium containing Ru(NH3)6(2+) and valinomycin, there was an uptake of K+ ions corresponding to 1.86 K+/e-. It is concluded that nearly 1 proton is translocated outwards through cytochrome oxidase per oxidizing equivalent injected in this medium. In low ionic strength sucrose-based medium, with Ru(NH3)6(2+) as reductant, delta H+O/e- was 1.05 in the presence of valinomycin, and -0.71 in the presence of FCCP. It is concluded that the translocation of protons is accompanied by net acid production in this medium.

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes by increasing the intramitochondrial concentration of Ca2+.

Increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase which result from the perfusion of rat hearts with adrenaline were still evident during the preparation of mitochondria in sucrose-based media containing EGTA (at 0 degrees C) and their subsequent incubation at 30 degrees C in Na+-free KCl-based media containing respiratory substrates and EGTA. The differences from control values gradually diminished with time of incubation, but were still present after 8 min. Similar increases resulting from an increase in the concentration of Ca2+ in the perfusing medium also persisted. However, similar increases caused by 5 mM-pyruvate were only maintained during the preparation of mitochondria, not their incubation. Parallel increases, within incubated mitochondria, were found in the activity of the 2-oxoglutarate dehydrogenase complex assayed at a non-saturating concentration of 2-oxoglutarate. The enhancement of the activities of both of these Ca2+-sensitive enzymes within incubated mitochondria as a result of perfusion with adrenaline or a raised concentration of Ca2+ in the medium could be abolished within 1 min by the presence of 10 mM-NaCl. This effect of Na+ was blocked by 300 microM-diltiazem, which has been shown to inhibit Na+-induced egress of Ca2+ from rabbit heart mitochondria [Vághy, Johnson, Matlib, Wang & Schwartz (1982) J. Biol. Chem. 257, 6000-6002]. The enhancements could also be abolished by increasing the extramitochondrial concentration of Ca2+ to a value where it caused maximal activation of the enzymes within control mitochondria. The results are consistent with the hypothesis that adrenaline activates rat heart pyruvate dehydrogenase by increasing the intramitochondrial concentration of Ca2+ and that this increase persists through to incubated mitochondria. Support for this conclusion was obtained by the yielding of a similar set of results from parallel experiments performed on control mitochondria that had firstly been preincubated (under conditions of steady-state Ca2+ cycling across the inner membrane) with sufficient proportions of Ca-EGTA buffers to achieve a similar degree of Ca2+-activation of pyruvate dehydrogenase (as caused by adrenaline) and had then undergone the isolation procedure again.

Varying effects of calcium on the oxidation of palmitate and α-ketoglutarate in isolated rat liver mitochondria incubated in KCl-based and sucrose-based media

1. 1. Isolated mitochondria from rat liver were incubated in the presence of [U- 14C]palmitate, ATP, CoA, carnitine, EGTA (ethylene glycol bis (β-aminoethyl ether) N, N′-tetraacetic acid) and varying amounts of calcium. 2. 2. When a KCl-based incubation medium was used, the oxidation of palmitate was inhibited when the concentration of free calcium was increased from about 0.1–10μM. 3. 3. When a sucrose-based incubation medium was used, the basal rate of palmitate oxidation was about half of that observed with the KCl-medium and calcium had a stimulatory effect. 4. 4. With the KCl-medium the rate of oxygen consumption was inhibited by calcium with α-ketoglutarate as well as palmitate as the respiratory substrate. 5. 5. No inhibitory effect of calcium was observed with succinate or β-hydroxybutyrate. 6. 6. With the KCl-medium and with α-ketoglutarate as the respiratory substrate, state 3 respiration but not state 4 respiration was inhibited by calcium. 7. 7. When the sucrose-medium was used, state 3 respiration was first inhibited by calcium, but this inhibition was gradually relieved and the respiratory rate finally became higher than it was before calcium addition.