L-malic-acid permeation in resting cells of anaerobically grown Saccharomyces cerevisiae

The study of permeation of L-malic acid in cells of Saccharomyces cerevisiae at pH 3.0 was carried out with (U-14C)-labelled L-malic acid. Resting cells were used in these experiments. They were previously anaerobically grown on glucose. This study showed that this transport is the result of two competitive mechanisms, one for the uptake and one for the efflux. The uptake mechanism seems to be a simple diffusion of the L-malic acid in a non-dissociated form. The efflux mechanism seems to be an active transport of L-malic acid that is very dependent on the temperature. At the steady state, the result of uptake and efflux mechanisms leads to an intracellular concentration which is twice or three times the extracellular concentration.     

pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase.

A pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase was carried out at 25 degrees C and 0.1 M ionic strength. The net charge on cytochrome c peroxidase due to proton association and dissociation varies from +32 at pH 2 to --50.2 at pH 12, while that of apocytochrome c peroxidase varies between +24.5 at pH 3 to --48 at pH 12. The apoprotein tented to aggregate below pH 3. Between pH 4 and 8, the titration behavior of both the native enzyme and the apoenzyme are consistent with the semi-empirical Linderstr?m-Lang theory. Between pH 9 and 12, the titration behavior of both the holo- and apoproteins suggest they assume a more extended conformation which reduces the electrostatic interaction charged groups on the surface. In the acid region, between pH 4 and 3, a similar transition occurs in which the protein expands 40% based on the electrostatic factor of the Linderstr?m-Lang theory.     

Engineering a prokaryotic apocytochrome c as an efficient substrate for Saccharomyces cerevisiae cytochrome c heme lyase

In spite of the extensive research using induced pluripotent stem (iPS) cells, the therapeutic potential of iPS cells in the treatment of peripheral nerve injury is largely unknown. In this study, we repaired peripheral nerve gaps in mice using tissue-engineered bioabsorbable nerve conduits coated with iPS cell-derived neurospheres. The secondary neurospheres derived from mouse iPS cells were suspended in each conduit (4000,000 cells per conduit) and cultured in the conduit in three-dimensional (3D) culture for 14 days. We then implanted them in the mouse sciatic nerve gaps (5 mm) (iPS group; n=10). The nerve conduit alone was implanted in the control group (n=10). After 4, 8 and 12 weeks, motor and sensory functional recovery in mice were significantly better in the iPS group. At 12 weeks, all the nerve conduits remained structurally stable without any collapse and histological analysis indicated axonal regeneration in the nerve conduits of both groups. However, the iPS group showed significantly more vigorous axonal regeneration. The bioabsorbable nerve conduits created by 3D-culture of iPS cell-derived neurospheres promoted regeneration of peripheral nerves and functional recovery in vivo. The combination of iPS cell technology and bioabsorbable nerve conduits shows potential as a future tool for the treatment of peripheral nerve defects.     

Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae

Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space. A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain. Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration. However, CCP1-disrupted cells were more sensitive to H₂O₂ than wild-type cells. We also constructed a CCP1–lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene. We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H₂O₂. These results hint that the biological function of CcP is to reduce H₂O₂ generated during aerobic respiratory process. Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.     

The biogenesis of mitochondria. II. The influence of medium composition on the cytology of anaerobically grown Saccharomyces cerevisiae

Yeast cells grown anaerobically have been shown to vary in their ultrastructure and absorption spectrum depending upon the composition of the growth medium. The changes observed in the anaerobically grown cells are governed by the availability of unsaturated fatty acids and ergosterol and a catabolite or glucose repression. All the cells contain nuclear and plasma membranes, but the extent of the occurrence of vacuolar and mitochondrial membranes varies greatly with the growth conditions. Cells grown anaerobically on the least nutritive medium, composed of 0.5% Difco yeast extract-5% glucose-inorganic salts (YE-G), appear to contain little vacuolar membrane and no clearly recognizable mitochondrial profiles. Cells grown anaerobically on the YE-G medium supplemented with Tween 80 and ergosterol contain clearly recognizable vacuolar membrane and some mitochondrial profiles, albeit rather poorly defined. Cells grown on YE-G medium supplemented only with Tween 80 are characterized by the presence of large amounts of cytoplasmic membrane in addition to vacuolar membrane and perhaps some primitive mitochondrial profiles. When galactose replaces glucose as the major carbon source in the medium, the mitochondrial profiles within the cytoplasm become more clearly recognizable and their number increases. In aerobically grown cells, the catabolite repression also operates to reduce the total number of mitochondrial profiles. The possibility is discussed that cells grown anaerobically on the YE-G medium may not contain mitochondrial membrane and, therefore, that such cells, on aeration, form mitochondrial membrane from nonmitochondrial sources. A wide variety of absorption compounds is observed in anaerobically grown cells which do not correspond to any of the classical aerobic yeast cytochromes. The number and relative proportions of these anaerobic compounds depend upon the composition of the growth medium, the most complex spectrum being found in cells grown in the absence of lipid supplements.     

The intramitochondrial location of cytochrome c peroxidase in wild-type and petite Saccharomyces cerevisiae

Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.     

Detection of inactive precursors of beta-glucanases in Saccharomyces cerevisiae

Accumulation and secretion of beta-glucanases have been studied in vivo by using a thermosensitive secretory mutant of Saccharomyces cerevisiae blocked at the endoplasmic reticulum level (sec 18-1). When incubated at the restrictive temperature no accumulation of active glucanases was observed. Following a shift to permissive conditions in the presence of cycloheximide a rise in the internal activity took place. The increase in total glucanase activity was partially due to the activation of an exo-glucanase that hydrolyzes PNPG. It is concluded that glucanases are synthesized in inactive precursor forms and are converted to the active forms in their secretory pathway.     

Subcellular fractionation by zonal centrifugation of glucose-repressed anaerobically grown Saccharomyces carlsbergensis

1. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucoserepressed Saccharomyces carlsbergensis, and the distributions of marker enzymes investigated after zonal centrifugation on sucrose gradients containing 2mm-MgCl(2). 2. These homogenates contained no detectable cytochrome c oxidase, succinate-cytochrome c oxidoreductase, succinate-ferricyanide oxidoreductase, l(+)-lactate-cytochrome c oxidoreductase or catalase. Cytochromes a+a(3) and c were not detected. 3. Zonal centrifugation of whole homogenates indicated complex density distributions of the sedimentable portions of NADH- and NADPH-cytochrome c oxidoreductases, adenosine triphosphatases (ATPases), adenosine pyrophosphatase (ADPase), pyrophosphatase and acid p-nitrophenyl phosphatase. Several different ATPases were distinguished on the basis of their sensitivities to oligomycin and ouabain. 4. Differential centrifugation of whole homogenates at 10(5)g-min left 80-90% of the protein, dithionite-reducible cytochrome b, acid hydrolases and pyrophosphatase in a supernatant (S(1)) together with 65 and 56% of the NADH- and NADPH-cytochrome c oxidoreductases respectively, 25% of the ATPases and 71% of the adenosine monophosphatase. 5. Further analysis of supernatant S(1) revealed the presence of a class of small particles containing NADPH-cytochrome c oxidoreductases and ATPases. 6. At least four different populations of large particles were distinguished. 7. Electron microscopy indicated that one of these corresponded to ;promitochondria' as described by other workers.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Precursor of beta-lactamase is enzymatically inactive. Accumulation of the preprotein in Saccharomyces cerevisiae

Synthesis and properties of the bacterial precursor of beta-lactamase (E.C.3.5.2.6) were studied in Saccharomyces cerevisiae transformants. A protease-deficient yeast mutant was transformed with the plasmid pADH040-2 conferring high expression of the bla gene. Besides precisely processed beta-lactamase, transformed yeast cells contained mainly bla precursor up to the amount of 2% of total cellular protein. The precursor was shown to be synthesized on free polysomes in vivo but could be processed with rough microsomal membranes in a cell-free translation system. By applying an isolation procedure using high-salt conditions, the labile precursor could be separated in a native form from the mature beta-lactamase. Thereby it could be shown that the pre-beta-lactamase had virtually no enzymatic activity in contrast to the mature enzyme, which was indistinguishable from bacterial beta-lactamase. Furthermore, the precursor was highly susceptible to proteolytic degradation by trypsin under conditions which did not affect the mature enzyme. Accordingly, the protein conformation of the precursor must be substantially different from that of the authentic beta-lactamase, demonstrating that specific processing and transport of beta-lactamase is associated with directing the protein to a distinct conformation.     

Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae.

Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database. We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product. The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes. Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type. Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation. The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p. The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power. Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase. However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant. Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase.