Mitochondrial number, cytochrome oxidase and succinic dehydrogenase activity in Xenopus laevis oocytes

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 395-409
Author(s):  
E. Marinos ◽  
F. S. Billett
Keyword(s):  
Xenopus Laevis ◽  
Cytochrome Oxidase ◽  
Specific Activity ◽  
Succinic Dehydrogenase ◽  
Mitochondrial Fraction ◽  
Xenopus Laevis Oocytes ◽  
Mitochondrial Fractions ◽  
Rate Of Increase ◽  
Mitochondrial Cloud ◽  
Mitochondrial Number

An estimate has been made of the numbers of mitochondria in the mitochondrial cloud (Balbiani body) of Xenopus laevis oocytes ranging in size from 50 to 250 μm. The mitochondrial number is expressed in terms of a ‘standard ’ organelle measuring 2 μm in length and 0·2 μm in diameter and is derived by measurements on electron micrographs of sections through the cloud. It is found that the amount of mitochondrial material rises very rapidly as the oocyte grows in size. At the time the cloud disperses, in oocytes of about 300 μm in diameter, it is estimated that there are the equivalent of over 500000 mitochondria in each cell. The rate of increase is very similar to the rate of accumulation of mitochondrial DNA during the same period of growth. Using a polarographic technique the specific activity of cytochrome oxidase and succinic dehydrogenase was determined in mitochondrial fractions isolated from oocytes over a size range of 80–1200 μm in diameter. Although the specific activity of succinic dehydrogenase remains constant that of cytochrome oxidase falls sharply during the period when the mitochondria are replicating rapidly, i.e. up to about 300 μm diameter. In larger oocytes the specific activity of enzymes appears to remain constant but increasing contamination of the isolated mitochondrial fraction does not allow conclusions to be drawn from the enzyme loading of the mitochondria once they have dispersed from the cloud. The results are discussed in relation to the possibility that mitochondrial replication preceeds, or at least outpaces, mitochondrial differentiation during the course of oogenesis.

scholarly journals Studies on bone enzymes. Distribution of acid hydrolases, alkaline phenylphosphatase, cytochrome oxidase and catalase in subcellular fraction of bone tissue homogenates

1965 ◽  
Vol 97 (2) ◽  
pp. 389-392 ◽  
Author(s):  
G Vaes ◽  
P Jacques
Keyword(s):  
Cytochrome Oxidase ◽  
Subcellular Fraction ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Distribution Patterns ◽  
Mitochondrial Fraction ◽  
Acid Hydrolases ◽  
Mitochondrial Fractions ◽  
Tissue Homogenates ◽  
Isopycnic Centrifugation

1. When bone homogenates were fractionated according to the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed except cytochrome oxidase were found to occur partly in soluble and partly in particulate fractions. Among the particle-bound enzymes, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome oxidase, in the microsomal fraction for alkaline phenylphosphatase and in the light-mitochondrial fraction for eight acid hydrolases and for catalase. 2. Combined heavy-mitochondrial and light-mitochondrial fractions were subfractionated by isopycnic centrifugation in density gradients of sucrose or glycogen. In the various systems tried, cytochrome oxidase showed a relatively narrow distribution range with a sharp peak; the acid hydrolases and catalase showed flat and irregular distribution patterns, differing slightly in shape from one enzyme to the other. However, it was not possible to achieve a marked separation between the various enzymes under study. 3. It is concluded from these results that the acid hydrolases belong to special cytoplasmic particles, probably lysosomes, and that these particles are physically and enzymically heterogeneous. Catalase appears to be non-mitochondrial and could also belong to the lysosomes; but the possibility of an association with another type of particle must be kept in mind in view of what is known of liver catalase. Alkaline phenylphosphatase is largely attached to microsomal elements.

Seasonal and starvation-induced changes in enzymatic pattern of frog nephron

1961 ◽  
Vol 201 (5) ◽  
pp. 781-785 ◽  
Author(s):  
Morris J. Karnovsky ◽  
S. Ralph Himmelhoch
Keyword(s):  
Cytochrome Oxidase ◽  
Enzyme Activities ◽  
Specific Activity ◽  
Hydrolytic Enzyme ◽  
Succinic Dehydrogenase ◽  
Krebs Cycle ◽  
Hexose Monophosphate ◽  
Hexose Monophosphate Shunt ◽  
Mitochondrial Fractions ◽  
Proximal Tubular

Oxidative and hydrolytic enzyme activities in the nephron of the frog were investigated using histochemical and spectrophotometric techniques. The distal tubule was found to possess high activities of enzymes of glycolysis and the Krebs cycle, as well as high cytochrome oxidase activity, but had only slight activities of the hexose-monophosphate shunt enzymes. The proximal tubule had high activities of glycolytic enzymes, enzymes of the hexose-monophosphate shunt, and enzymes of the Krebs cycle, except for succinic dehydrogenase, which was present in very low activity. Proximal tubular cytochrome oxidase was found to vary according both to the season and to environ mental conditions. In freshly caught summer frogs it was present in high activity, but in winter frogs or summer frogs starved for periods of 10–30 days, no proximal tubular cytochrome oxidase could be detected, although other proximal tubular enzyme activities remained unchanged as far as could be histochemically determined. The specific activity of cytochrome oxidase in mitochondrial fractions was found to decrease by about 50% in the kidneys of starved and winter frogs, as compared with summer frogs.

scholarly journals MORPHOLOGICAL AND BIOCHEMICAL CHANGES IN RAT SYNAPTOSOME FRACTIONS DURING NEONATAL DEVELOPMENT

1971 ◽  
Vol 51 (2) ◽  
pp. 484-498 ◽  
Author(s):  
Nicholas K. Gonatas ◽  
Lucila Autilio-Gambetti ◽  
Pierluigi Gambetti ◽  
Brenda Shafer
Keyword(s):  
Postnatal Development ◽  
Adult Animal ◽  
Specific Activity ◽  
Brain Homogenate ◽  
Subcellular Fractions ◽  
Presynaptic Terminals ◽  
Adult Rats ◽  
Total Brain ◽  
Mitochondrial Fractions ◽  
Rate Of Increase

A biochemical and quantitative morphologic study of presynaptic endings during postnatal development was carried out in subcellular fractions from cerebral cortex of 1, 4, 8, 12, and 18 day old and adult rats. Crude mitochondrial fractions were subfractionated in Ficoll gradients and all resulting fractions were examined in the electron microscope. Presynaptic terminals and other intact processes were counted. Protein content and enzyme activities were assayed in the fractions and in total brain homogenate. In the first and fourth day of life, most of the presynaptic terminals were found in two "light" fractions, between supernatant and 7.5% Ficoll, where they accounted, respectively, for 6 and 22% of all the processes. Progressively with age, more presynaptic terminals were found in the traditional "synaptosomal" fractions between 7.5 and 13% Ficoll. In that region of the gradient, 40, 54, 75, and 89% of the processes were presynaptic endings at 8, 12, and 18 postnatal days and in the adult animal, respectively. A similar shift from the lighter to the heavier fractions was observed in the distribution of choline acetyltransferase and acetylcholinesterase between days 8 and 12. The rate of increase of the specific activity of these two enzymes paralleled that of the percentage of the presynaptic endings after day 8. This study indicates that subcellular fractions can be used to study formation and maturation of synapses during postnatal development.

scholarly journals Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5′[γ-thio]triphosphate

1996 ◽  
Vol 316 (3) ◽  
pp. 793-803 ◽  
Author(s):  
Ling LAN ◽  
Michael J. BAWDEN ◽  
Amanda M. AULD ◽  
Greg J. BARRITT
Keyword(s):  
Xenopus Laevis ◽  
Divalent Cation ◽  
Initial Rate ◽  
Regulatory Protein ◽  
Xenopus Laevis Oocytes ◽  
High Concentration ◽  
Basal Rate ◽  
Rate Of Increase ◽  
Dependent Protein ◽  
Ca2 Channels

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631–642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]o) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]o. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 μM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]i and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281–309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5´-[β-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes; (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

High tyrosinase activity in albino Xenopus laevis oocytes

Development ◽  
1976 ◽  
Vol 36 (3) ◽  
pp. 555-559
Author(s):  
A. H. Wyllie ◽  
E. M. De Robertis
Keyword(s):  
Molecular Weight ◽  
Xenopus Laevis ◽  
Specific Activity ◽  
High Specific Activity ◽  
Low Molecular Weight ◽  
Xenopus Laevis Oocytes ◽  
Melanin Synthesis ◽  
Wild Type ◽  
Albino Mutant

Tyrosinase was measured in oocytes of the recently described albino mutant (avav) of Xenopus laevis. Although these oocytes show no pigmentation and the eggs are known to contain no melanosomes, tyrosinase — which is probably the only enzyme necessary for melanin synthesis from tyrosine — was increased more than twofold relative to the wild type. Tyrosinase recovered from albino and wild type oocytes showed the same KM with respect to tyrosine, and this was not altered by previous gonadotrophin stimulation in vivo. The tyrosi-nase assay, based on [14]tyrosine incorporation into acid-insoluble products, was of greater sensitivity than previously described methods of the same type, through removal of low molecular weight material from the oocyte homogenate prior to incubation, and the use of tyrosine of high specific activity.

scholarly journals Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum-mitochondrion complex in rat liver

1982 ◽  
Vol 208 (2) ◽  
pp. 505-507 ◽  
Author(s):  
S Parimoo ◽  
N Rao ◽  
G Padmanaban
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Specific Activity ◽  
Mitochondrial Fraction

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum-mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum-mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes

Zygote ◽  
2011 ◽  
Vol 20 (3) ◽  
pp. 237-242 ◽  
Author(s):  
M.C. Vaccaro ◽  
M. Wilding ◽  
B. Dale ◽  
C. Campanella ◽  
R. Carotenuto
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Stage I ◽  
Xenopus Laevis Oocytes ◽  
Cytoskeletal Network ◽  
Vegetal Cortex ◽  
Vegetal Pole ◽  
Mitochondrial Cloud ◽  
Germinal Plasm

SummaryIn Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I–II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II–III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

scholarly journals Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion

1996 ◽  
Vol 319 (3) ◽  
pp. 755-760 ◽  
Author(s):  
Roland B GREGORY ◽  
Greg J BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Xenopus Laevis ◽  
Membrane Fusion ◽  
Direct Interaction ◽  
Protein S ◽  
Detectable Effect ◽  
Xenopus Laevis Oocytes ◽  
Endoplasmic Reticulum Membrane ◽  
Rate Of Increase

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro-Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+o-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trpl cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2–17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5´-[γ-thio]triphosphate (GTP[S]), guanosine 5´-[β,γ-imido]triphosphate (p[NH]ppG) and AlF4-, but not guanosine 5´-[β-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] or AlF4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.

Sign in / Sign up

Export Citation Format

Share Document