Carmen G. Vallejo

Animal mitochondrial biogenesis and function: a regulatory cross-talk between two genomes

Mitochondria play a pivotal role in cell physiology, producing the cellular energy and other essential metabolites as well as controlling apoptosis by integrating numerous death signals. The biogenesis of the oxidative phosphorylation system (OXPHOS) depends on the coordinated expression of two genomes, nuclear and mitochondrial. As a consequence, the control of mitochondrial biogenesis and function depends on extremely complex processes that require a variety of well orchestrated regulatory mechanisms. It is now clear that in order to provide cells with the correct number of structural and functional differentiated mitochondria, a variety of intracellular and extracellular signals including hormones and environmental stimuli need to be integrated. During the last few years a considerable effort has been devoted to study the factors that regulate mtDNA replication and transcription as well as the expression of nuclear-encoded mitochondrial genes in physiological and pathological conditions. Although still in their infancy, these studies are starting to provide the molecular basis that will allow to understand the mechanisms involved in the nucleo-mitochondrial communication, a cross-talk essential for cell life and death.

Drosophila cathepsin B-like proteinase: A suggested role in yolk degradation

A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.

Interferences by sulfhydryl, disulfide reagents and potassium ions on protein determination by Lowry's method

Abstract Sulfhydryl, disulfide reagents, and also potassium ions interfere with the protein determination by Lowrys method. The concentrations of these substances which can be used without serious interference are given. Sulfhydryl and disulfide reagents yield a color with the same absorption spectrum as that caused by proteins. At moderate concentrations this interference can be overcome by running appropriate blanks. Potassium ions produce a white precipitate that can be removed without change of color intensity by centrifuging the reaction mixtures after color development.

Dinucleosidasetetraphosphatase in rat liver and Artemia salina.

A comparative study of an enzymatic activity present in Artemia salina and rat liver which specifically splits dinucleoside tetraphosphates is presented. All the purine and pyrimidine dinucleoside tetraphosphates tested, i.e. diadenosine, diguanosine, dixanthosine and diuridine tetraphosphates, were substrates of both enzymes with similar maximum velocities and Km values, (around 10 muM). The inhibition by nucleotides of the enzyme from the two sources is also similar. Particularly relevant is the strong inhibition caused by nucleoside tetraphosphates which have Ki values in the nanomolar range. The Artemia enzyme has a slightly lower molecular weight (17 500) than the liver enzyme (21 000) and is more resistant to acidic pH. Based on previous findings, the enzyme from Artemia salina was named diguanosinetetraphosphatase (EC 3.6.1.17) by the Enzyme Commission. The results presented in this paper show that the liver and Artemia enzymes are similar, and we propose to name this enzyme as dinucleosidetetraphosphatase or dinucleoside-tetraphosphate nucleotidehydrolase.

Diguanosinetetraphosphate guanylohydrolase in Artemia salina

Abstract 1. 1. The diguanosinetetraphosphate guanylohydrolase (EC 3.6.1.17) of Artemia salina has been found to be located in the cytosol while its substrate, diguanosine tetraphosphate, is in the 700 × g sediment. 2. 2. Two spectrophotometric methods have been developed to study this enzyme. One is based in the evaluation of one of the products, GTP, coupled to the auxiliary enzymes phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase. The other method is based on the hyperchromicity observed at 252 nm by the splitting of diguanosine tetraphosphate. 3. 3. With a partially purified preparation, the following enzymatic properties have been found: K m for diguanosine tetraphosphate, 5 μM. GMP, GDP, GTP and ATP were competitive inhibitors of the reaction with K i values of 24, 56, 14 and 30 μM respectively. 4. 4. Even lower values were obtained with the guanosine 5′-tetraphosphate and adenosine 5′-tetraphosphate, which were 0.006 and 0.13 μM, respectively. These rather low K i values suggest a possible role for these compounds as metabolic regulators.

The Maternal Origin of Acid Hydrolases in Drosophila and Their Relation with Yolk Degradation

The acid hydrolases of Drosophila are of maternal origin and appear subjected to differentiated control during embryogenesis. The enzymes are found associated with yolk granules. This association decreases during embryogenesis, in parallel with yolk degradation. As suggested before (Medina et al. Arch. Biochem. Biophys., 263, 355–363) the acid proteinase seems to be involved in the degradation of the yolk protein. The developmental profile of activity of the proteinase fits rather well with its involvement in the degradation of yolk granules. We have isolated intermediates of degradation of these subcellular structures. The intermediates have acid hydrolase activity and decrease in buoyant density during embryogenesis, in parallel with yolk degradation. The electron microscopic analysis has revealed that they are morphologically heterogenuous. A population of yolk granules appears to store mitochondria in their interior. The mitochondrial marker cytochrome oxidase is detected in density gradients associated with the intermediates of degradation, also supporting the storage of mitochondria in yolk granules in early development. The fact that the acid hydrolases are of maternal origin suggests that they have a role during embryogenesis. We propose that acid hydrolase(s) are involved in yolk degradation.

Acid hydrolases during Artemia development: A role in yolk degradation

Abstract 1. 1. A broad group of acid hydrolytic activities have been determined during Artemia development including cathepsin B acid ribonuclease, acid deoxyribonuclease, acid phosphatase, acid phosphodiesterase, β-glucosidase, β-N-acetylgalactosaminidase and acid lipase. These enzymes present maximum activity in nauplii, when yolk degradation is maximum. 2. 2. Artemia cathepsin B proteinase is able to degrade lipovitellin in vitro with a pattern similar to that found in vivo. These results suggest the involvement of acid hydrolases in the degradation of yolk. 3. 3. In cryptobiotic embryos, acid hydrolases were found associated with structures of buoyant density 1.18. The structures appeared at the electron microscope as 0.4 μm dia. vesicles when revealed by acid phosphatase cytochemistry.

Thyroid hormone increases transcription of GA‐binding protein/nuclear respiratory factor‐2 α‐subunit in rat liver

Thyroid hormone (TH) regulates mitochondrial respiratory rate by activating coordinated transcription in the nucleus and mitochondria. Whereas TH activates transcription of mitochondrial genes directly, the activation of nuclear‐encoded mitochondrial genes is probably executed by indirect unknown mechanisms. Nuclear respiratory factors (NRF)‐1 and GA‐binding protein (BP)/NRF‐2 may function as transacting genes, but regulation of these genes by TH is not demonstrated. We show that TH administration to hypothyroid rats promptly increases GABP/NRF‐2 α‐subunit mRNA levels in the liver, without significant changes in β, γ subunits. In run‐on and time‐course experiments, the transcription rate and protein levels increased three‐fold in response to TH, indicating GABP/NRF‐2 transcriptional regulation. The results also support the notion that ATP synthase β‐subunit is regulated by TH through the indirect activation of GABP/NRF‐2.

The trypsin-like proteinase of Artemia: Yolk localization and developmental activation

Abstract 1. 1. A trypsin-like proteinase has been found in Artemia dormant embryos localized in yolk granules associated to lipovitellin. 2. 2. During development, and in parallel with an exponential increase in the activity of the proteinase, we have observed (a) gradual changes in its subcellular localization from the yolk granules to the cytosol, (b) a gradual decrease in the molecular weight of the cytosolic enzyme, and (c) a gradual improvement in the accessibility of substrates and inhibitors to the proteinase active centre. 3. 3. The partially purified cytosolic proteinase reacted with anti-lipovitellin antiserum. The results are discussed in relation to the yolk origin of the proteinase and its activation during development.

Degradation of yolk in the brine shrimp Artemia. Biochemical and morphological studies on the involvement of the lysosomal system

The degradation of yolk granules during the development of Artemia was studied. The results obtained suggest that lysosomes are involved in the process. In homogenates of embryos and larvae at different stages of development, the distribution of 2 lysosomal markers, acid phosphatase and cathepsin B, was studied by sucrose isopycnic gradient centrifugation. Three peaks of enzyme activity of densities > 1.3 and around 1.25 and 1.18 were observed. As revealed by electron microscope analysis, the 3 peaks were found to be associated with increasingly degraded yolk structures which stained for acid phosphatase. The process can be mimicked in vitro by incubating isolated yolk granules and lysosomes. The enzyme activity levels of the 3 peaks observed during development presented an oscillatory pattern, suggesting that degradation of yolk is cyclic. Five cycles of degradation were observed during the initial 60 hr of development.