Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.     

Mechanism of reductive activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance spectroelectrochemical study

The mechanism of reductive methylation of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate:homocysteine methyltransferase, EC 2.1.1.13) has been investigated by electron paramagnetic resonance (EPR) spectroelectrochemistry. The enzyme as isolated is inactive, and its UV/visible absorbance and EPR spectra are characteristic of cob(II)alamin. There is an absolute requirement for catalytic amounts of AdoMet and a reducing system for the formation and maintenance of active enzyme during in vitro turnover. The midpoint potentials of the enzyme-bound cob(II)alamin/cob(I)alamin and cob(III)alamin/cob(II)alamin couples have been determined to be -526 +/- 5 and +273 +/- 4 mV (versus the standard hydrogen electrode), respectively. The presence of either CH3-H4folate or AdoMet shifts the equilibrium distribution of cobalamin species observed during reduction by converting cob(I)alamin to methylcobalamin. The magnitude of these shifts is however vastly different, with AdoMet lowering the concentration of cob(II)alamin at equilibrium by a factor of at least 3 X 10(7), while CH3-H4folate lowers it by a factor of 19. These studies of coupled reduction/methylation reactions elucidate the absolute requirement for AdoMet in the in vitro assay system, in which the ambient potential is approximately -350 mV versus the standard hydrogen electrode. At this potential, the equilibrium distribution of cobalamin in the presence of CH3-H4folate would be greatly in favor of the cob(II)alamin species, whereas in the presence of AdoMet the equilibrium favors methylated enzyme. In these studies, a base-on form of cob(II)alamin in which the dimethylbenzimidazole substituent of the corrin ring is the lower axial ligand for the cobalt has been observed for the first time on methionine synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlled potential enzymology of methyl transfer reactions involved in acetyl-CoA synthesis by CO dehydrogenase and the corrinoid/iron-sulfur protein from Clostridium thermoaceticum

Many anaerobic bacteria fix CO2 via the Wood pathway of acetyl-CoA synthesis. Carbon monoxide dehydrogenase (CODH), also called acetyl-CoA synthase, accepts the methyl group from the methylated corrinoid/iron-sulfur protein (C/Fe-SP), binds a carbonyl group from CO, CO2, or the carboxyl of pyruvate, and binds coenzyme A. Then CODH catalyzes the synthesis of acetyl-CoA from these enzyme-bound groups. Here, we have characterized the methyl transfer steps involved in acetyl-CoA synthesis. We have studied the reactions leading to methylation of CODH by methyl iodide and shown an absolute requirement of the C/Fe-SP in this reaction. In addition, we have discovered and partly characterized two previously unknown exchange reactions catalyzed by CODH: between the methylated C/Fe-SP and methylated CODH and between methylated CODH and the methyl moiety of acetyl-CoA. We have performed these two exchange reactions, methylation of the C/Fe-SP, and methylation of CODH at controlled potentials. The rates of all these reactions except the exchange between methylated C/Fe-SP and methylated CODH are accelerated (from 1 to 2 orders of magnitude) when run at low potentials. Our results provide strong evidence for a nucleophilic redox-active metal center on CODH as the initial acceptor of the methyl group from the methylated C/Fe-SP. This metal center also is proposed to be involved in the cleavage of acetyl-CoA in the reverse reaction.     

N-acetyl-p-benzoquinone imine-induced changes in the energy metabolism in hepatocytes

The effect of N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, on the energy metabolism in isolated hepatocytes was investigated. Incubation of cells with NAPQI (400 microM) resulted in an immediate uptake into the mitochondria, followed by both reduction and glutathione conjugation of the quinone imine. These reactions were extremely rapid and were associated with depletion of the mitochondrial ATP content (greater than 80% depletion after 1 min exposure). The loss of ATP was accompanied by increases in ADP and AMP, as well as NADP. No effect on mitochondrial NAD was observed during this initial phase. Similar alterations were produced by NAPQI in the cytosolic compartment. Furthermore, incubation of hepatocytes with NAPQI inhibited oxygen consumption by nearly 90% within 10 s. In parallel to these biochemical changes, there was marked bleb formation on the surface of the hepatocytes, which was found to precede cell death (trypan blue uptake). In conclusion, our results demonstrate that during exposure of hepatocytes to NAPQI, dramatic changes in cellular energy metabolism occur. These biochemical alterations may be caused by a rapid decrease in mitochondrial function, and they may play an important role in the initiation of NAPQI-induced cytotoxicity.     

The guanosine 3',5'-bis(diphosphate) (ppGpp) cycle. Comparison of synthesis and degradation of guanosine 3',5'-bis(diphosphate) in various bacterial systems.

4-(3-Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl-14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme-coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C-labelled coenzyme analogue and subseqeuntly carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys-38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys-43 in yeast alcohol dehydrogenase and Cys-46 in the horse liver enzyme but, unlike the latter two, Cys-38 is not reactive towards iodoacetate in the native bacterial enzyme.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Predation by squirrel monkeys and double-toothed kites on tent-making bats

Central American squirrel monkeys (Saimiri oerstedi) appear to recognize the modified leaves that phyllostomid bats utilize for diurnal roost sites. The monkeys visually and manually search these bat tents for both bats and insects. Adult males are the most successful at capturing bats. Nonvolant juvenile bats are more vulnerable to monkey predation than are adults. Bats that escape monkey predation frequently are captured by doubletoothed kites (Harpagus bidentatus) that tend foraging troops of monkeys. Predation by squirrel monkeys, coupled with that of double-toothed kites, may be a significant source of mortality for tent-making bats.     

Epithelial polarity and spindle orientation: intersecting pathways

During asymmetric stem cell divisions, the mitotic spindle must be correctly oriented and positioned with respect to the axis of cell polarity to ensure that cell fate determinants are appropriately segregated into only one daughter cell. By contrast, epithelial cells divide symmetrically and orient their mitotic spindles perpendicular to the main apical–basal polarity axis, so that both daughter cells remain within the epithelium. Work in the past 20 years has defined a core ternary complex consisting of Pins, Mud and Gαi that participates in spindle orientation in both asymmetric and symmetric divisions. As additional factors that interact with this complex continue to be identified, a theme has emerged: there is substantial overlap between the mechanisms that orient the spindle and those that establish and maintain apical–basal polarity in epithelial cells. In this review, we examine several factors implicated in both processes, namely Canoe, Bazooka, aPKC and Discs large, and consider the implications of this work on how the spindle is oriented during epithelial cell divisions.