Mechanism of polyamine spermidine uptake by Xenopus laevis oocytes

In this study, the mechanisms of polyamine spermidine (Spd) uptake were investigated in Xenopus laevis oocytes. Spd uptake followed a sigmoidal kinetics with [S]90/[S]10 = 3 microM and Hill interaction coefficient (n) = 2. The order of magnitude of uptake and efflux was similar (t1/2 = 45 min). The equilibrium potential for Spd, calculated by Nenrst equation, was 90.78 mV. Free energy change for the uptake (delta G) was found to be 2.31 Kcal/mole of Spd. During efflux, Spd was not converted into putrescine or spermine. It seems that there are two types of Spd uptake pathways: Na(+)-dependent and Na(+)-independent since replacement of Na+ from incubation medium did not completely abolish the Spd uptake. The Na(+)-dependent component of Spd uptake was shared neither by system A nor by system ASC amino acids.     

Characterization of DNA-protein complexes from the mitochondria of Xenopus laevis oocytes

Mitochondrial DNA (mtDNA)-protein complexes (nucleoids) from Xenopus laevis oocytes were purified either on rate-zonal sucrose or isopyknic metrizamide gradients. From electron microscopic studies and staphylococcal nuclease digestion experiments mtDNA appears to be packaged into regular beaded structures. Protein electrophoretic analysis and M banding results show that mtDNA is associated with the membrane structures and also with few specific proteins including one acid-soluble polypeptide of 28 kD.     

In vivo measurements of control coefficients for hexokinase and glucose-6-phosphate dehydrogenase in Xenopus laevis oocytes

Hexokinase and glucose-6-phosphate dehydrogenase activities were increased in Xenopus laevis oocytes by microinjection of commercial pure enzymes. The effect of increased fractional activities on glycogen synthesis or on the production of 14CO(2) (the oxidative portion of the pentose phosphate pathway) was investigated by microinjection of [1-(14)C]glucose and measurements of the radioactivity in glycogen and CO(2). Control coefficients calculated from the data show that hexokinase plays an important role in the control of glycogen synthesis (control coefficient=0.7) but its influence on the control of the pentose phosphate pathway is almost nil (control coefficient=-0.01). Glucose-6-phosphate dehydrogenase injections did not affect the production of 14CO(2) by the pentose phosphate pathway, indicating that other factors control the operation of this pathway. In addition, an almost null control of this enzyme on glycogen synthesis flux was observed.     

The number of mitochondria in Xenopus laevis ovulated oocytes

An attempt was made to estimate the total number of mitochondria in Xenopus laevis ovulated oocytes. For this purpose the necessary basic parameters were calculated employing planimetry and simple mathematical formulas. It was found that the number of mitochondria in the ovulated oocyte of Xenopus is of the order of 10(7). The significance of this finding is discussed.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A⁺-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.     

Evidence for a mitochondrial chromosome in Xenopus laevis oocytes

When Xenopus laevis mitochondria are gently lysed at physiologic ionic strength, mitochondrial DNA (mitDNA) is extracted associated with proteins. Sedimentation and buoyant density studies indicate that proteins are bound to mitDNA at a ratio of about 1/1. This DNA-protein complex visualized by electron microscopy after fixation with glutaraldehyde appears as a relaxed circular molecule consisting of an average of 48 globular particles interconnected by a thin filament.     

Heterogeneous distribution and replication activity of mitochondria in Xenopus laevis oocytes

In early diplotene oocytes of Xenopus laevis mitochondria are not dispersed all over the cytoplasm but gathered in a well described mitochondrial mass [18]. When tracing these organelles during active vitellogenesis we observe that some of them are involved in the elaboration of a cortical layer at the vegetative hemisphere of the cell while others stay around the nucleus. The latter contribute to the transient formation of a mitochondrial crown throughout active mitochondriogenesis. Autoradiographic studies of thymidine incorporation into mtDNA suggest a differential participation of each organelle to the final population of a full-grown oocyte according to its position in the cytoplasm.     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively     

In vivo synthesis and turnover of cytoplasmic ribosomal RNA by stage 6 oocytes of Xenopus laevis.

Cytoplasmic ribosomal RNA (rRNA) synthesis was detected in white-banded stage 6 oocytes taken from female Xenopus laevis which were injected with [³H]guanosine 7 days previously. The specific radioactivity of the rRNA in oocytes collected from injected females by weekly laparotomies displays first-order exponential decay. Calculated values for the half-life of rRNA ranged from 9.1–30.9 days in experiments on four animals. The concept of ribosomes in large ovarian oocytes of amphibians as an absolutely stable, long-term storage product appears incorrect.     

Subcellular in vivo 1H MR spectroscopy of Xenopus laevis oocytes

In vivo magnetic resonance (MR) spectra are typically obtained from voxels whose spatial dimensions far exceed those of the cells they contain. This study was designed to evaluate the potential of localized MR spectroscopy to investigate subcellular phenomena. Using a high magnetic field and a home-built microscopy probe with large gradient field strengths, we achieved voxel sizes of (180 microm)3. In the large oocytes of the frog Xenopus laevis, this was small enough to allow the recording of the first compartment-selective in vivo MR spectra from the animal and vegetal cytoplasm as well as the nucleus. The two cytoplasmic regions differed in their lipid contents and NMR lineshape characteristics-differences that are not detectable with whole-cell NMR techniques. In the nucleus, the signal appeared to be dominated by water, whereas other contributions were negligible. We also used localized spectroscopy to monitor the uptake of diminazene acturate, an antitrypanosomal agent, into compartments of a single living oocyte. The resulting spectra from the nucleus and cytoplasm revealed different uptake kinetics for the two components of the drug and demonstrate that MR technology is on the verge of becoming a tool for cell biology.     

Synthesis of the mitochondrial inner membrane in cultured Xenopus laevis oocytes.

The purpose of this study was to investigate the contribution of mitochondrial and cytoplasmic protein synthesis to the biogenesis of cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase EC 1.9.3.1) and rutamycin-sensitive adenosine triphosphatase (ATP phosphohydrolase EC 3.6.1.3) in cultured oocytes of the toad, Xenopus laevis. X. laevis cytochrome oxidase was purified over 23-fold with respect to specific activity and over 29-fold with respect to specific heme a content from oocyte submitochondrial particles. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate separated the enzyme into six subunits with molecular weights of 44,000, 33,000, 23,000, 17,000, 12,000 and 9,500. the synthesis of the three larger subunits is sensitive to chloramphenicol (an inhibitor of mitochondrial protein synthesis), indicating that these subunits are made on mitochondrial ribosomes; the synthesis of the three smaller subunits is sensitive to cycloheximide (an inhibitor of cytoplasmic protein synthesis) and therefore occurs on cytoplasmic ribosomes. X. laevis rutamycin-sensitive ATPase, purified over 19-fold from oocyte submitochondrial pparticles, consists of 10 subunits with molecular weights of 56,000, 53,000, 41,000, 32,000, 29,000, 24,000, 21,000, 17,500 (2), and 11,500 on sodium dodecyl sulfate-polyacrylamide gels. The 29,000, 21,000, and one of the 17,500-dalton polypeptides are synthesized in the presence of cycloheximide and are, therefore, products of mitochondrial protein synthesis; the synthesis of the remaining seven subunits occurs in the presence of chloramphenicol, indicating that these subunits are made on cytoplasmic ribosomes. The synthesis of protein by mitochondria in cultured oocytes appears to be dependent upon cytoplasmic protein synthesis. In the presence of cycloheximide, the mitoribosomal synthesis of the subunits of cytochrome oxidase and rutamycin-sensitive ATPase is detectable only after a prior inhibition of mitochondrial protein synthesis by chloramphenicol. Oocyte mitochondrial ribosomes synthesize at least nine polypeptides after chloramphenicol treatment, three of which are components of neither cytochrome oxidase nor rutamycin-sensitive ATPase.     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Dissociation of mitochondrial malate dehydrogenase

The kinetics of the dissociation reaction under acidic conditions of the dimeric pig and chicken mitochondrial malate dehydrogenases (EC 1.1.1.37) have been studied. The dissociation of the pig enzyme is completely reversible. The pK for dissociation determined by light-scattering measurements agrees within experimental error with the pK value of 5.25 measured for a tyrosine-carboxylate pair. The rate constants for the dissociation reaction and for the protonation process of this tyrosine are in close agreement. Thus, the tyrosine-carboxylate pair can be used as indicator of the dissociation reaction. The dissociation of the chicken enzyme proceeds around pH 4.5 at a much lower rate. A true equilibrium between dimer and monomers is not found, since the monomer gradually unfolds at this pH. The monomers of both enzymes, pig and chicken mitochondrial malate dehydrogenase, show the same stability towards acid. The difference in stability of the dimeric forms, therefore, must be due to an altered subunit contact area.