Artemia Embryos Research Articles

Adenosine deaminase isozymes in Artemia

1. 1. Three isozymes of adenosine deaminase, termed II, III and IV, have been detected in Artemia embryos. 2. 2. Their pI values, determined by chromatofocusing, were 4.9, 5.0 and 5.2, respectively. 3. 3. Upon development to larvae, a different isozyme (I) is induced, with a pI value of 4.2 as determined by isoelectric focusing.

Diadenosine 5',5'"-P1,P4-tetraphosphate in developing embryos of Artemia.

Diadenosine 5',5'"-P1,P4-tetraphosphate (Ap4A) has been detected in cysts and developing embryos of the brine shrimp Artemia in amounts 10(4)-10(6) times lower than that of the guanine analogue, Gp4G. The unexpectedly high level of Ap4A in dormant cysts of 2.37 pmol/10(6) cells can be reduced to 0.03 pmol/10(6) cells by decapsulation and storage in saturated NaCl. When development is reinitiated, the Ap4A content of the decapsulated embryos undergoes a rapid 125 -fold increase, reaching a maximum of 3.79 pmol/10(6) cells at the point of emergence when DNA replication begins. If replication is delayed by hypoxia, the Ap4A level is adjusted in order to reach the same maximum value when replication finally begins. As replication proceeds, the level of Ap4A declines again. Unlike mammalian cells, Ap4A in Artemia is less metabolically labile than ATP. These results are consistent with the suggested role of Ap4A in the initiation of DNA synthesis.

Adenylosuccinate lyase from Artemia embryos. Purification and properties.

In crude extracts, the molecular form of adenylosuccinate lyase is pH-dependent as studied by gel filtration and sucrose gradient centrifugation. At pH values of 8.7 and 6.5, the enzyme exhibits molecular forms of 200 kDa and larger than 500 kDa, respectively. At pH values of 7.0-7.5, forms of intermediate molecular weight were detected. Interconversion among the different molecular forms of adenylosuccinate lyase was not achieved when the enzyme was subjected to two successive chromatographic steps on Sepharose CL-6B using elution buffers at two different pH values. A unique form of 200 kDa was observed, regardless of the pH of the buffer used, upon either gel filtration or sucrose gradient centrifugation in the presence of 1 M NaCl. The enzyme from Artemia cysts was purified to homogeneity. It had molecular mass of 200 kDa and gave a single band of 56 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km values for adenylosuccinate, AMP, and fumarate were 1, 36, and 350 microM, respectively. The enzyme exhibits a Uni Bi-ordered mechanism and is maximally active at a pH value of 8.0-8.5. For maximum activity, the enzyme requires an ionic strength equivalent to 80 mM KCI. The isoelectric point determined by chromatofocusing was 5.04.

Distribution of elongation factor 2 between particulate and soluble fractions of the brine shrimp Artemia during early development.

The ADP-ribosylation of elongation factor 2 (EF-2) in vitro was used to quantitate EF-2 and to determine its subcellular distribution in extracts of Artemia embryos at different stages of development. In extracts from dormant cysts of Artemia 40-45% of EF-2 is complexed to macromolecules smaller than ribosomes, whereas the remainder is soluble or free in the cytosol. During early development the amount of "complexed" EF-2 decreases markedly concomitant with an increase in the pool of soluble EF-2. Complexed EF-2 was found to be associated with macromolecules which sediment at 16S-20S and 40S-50S and not with monoribosomes or polyribosomes as reported for mammalian systems. The data show that the decrease in complexed EF-2 is associated with the resumption of development in Artemia.

Protein synthesis in brine shrimp embryos and rabbit reticulocytes. The effect of Mg2+ on binary (eukaryotic initiation factor 2 X GDP) and ternary (eukaryotic initiation factor 2 X GTP X met-tRNAf) complex formation.

We have prepared eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes and Artemia embryos and studied the effect of Mg2+ on binary (eIF-2 X GDP) and ternary (eIF-2 X GTP X Met-tRNAf) complex formation. Under conditions where Mg2+ inhibits Met-tRNAf binding to reticulocyte eIF-2, ternary complex formation with Artemia eIF-2 is not inhibited. Similarly, the formation of eIF-2 X GDP with Artemia eIF-2 is stimulated by Mg2+, whereas the corresponding reticulocyte binary complex is strongly inhibited. In the presence of 1 mM Mg2+, the isolated Artemia eIF-2 X GDP complex is stable in the absence of any added nucleotide, but readily exchanges bound GDP for free GTP. However, the reticulocyte eIF-2 X GDP complex is significantly more stable in the presence of GTP, and nucleotide exchange is dependent upon the addition of a factor isolated from either the postribosomal supernatant or the high salt wash of rabbit reticulocyte ribosomes. This factor also stimulates Met-tRNAf binding to both Artemia and reticulocyte eIF-2.

Presence of two isozymes of adenylosuccinate synthetase in Artemia salina embryos. Purification and properties

1. 1. Two isozymes of adenylosuccinate synthetase, termed I and II have been separated by chromatography on DEAE-cellulose (DE-52), in Artemia embryos. 2. 2. The following data refer to form I and II: apparent molecular weights determined by sucrose gradient centrifugation and chromatography on Sephadex G-100: (I) 96,700, 90,000 and (II) 97,800, 83,000; K m values for their three substrates IMP, GTP and aspartate: (I) 26, 29, 600 and (II) 20, 25, 700 μM; fructose 1,6-biphosphate was a competitive inhibitor towards IMP with a K i value of 22 μM for both isozymes. 3. 3. The relationship between the Artemia enzyme and that from rat liver and other sources, is also discussed in this paper.

Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo 31P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.

Analysis of the membrane-associated poly(A) +RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization : Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm 3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A) +RNA was therefore investigated. In gel electrophoresis poly(A) +RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3·10 5 and 5·10 5 Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A) +RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A) +RNA component of approx. 5·10 5 Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A) +RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A) +RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A) +RNA from developing embryos. The hybridization of labelled, nuclear poly(A) +RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A) +RNA in dormant cysts is essentially of nuclear origin. The 5·10 5-Da component is largely homologous with the corresponding component of nuclear poly(A) +RNA at later stages.

Purification and properties of a polyadenylate polymerase from Artemia dormant embryos

Soluble extracts from encysted dormant embryos of Artemia contain a poly(A) polymerase activity which has been partially purified and characterized. The enzyme requires manganese, an RNA primer and ATP for maximal activity. The K m for ATP is 0.04 mM and the enzyme is inhibited by concentrations higher than 0.5 mM ATP. dATP replaces ATP with a 15% efficiency. The K m for dATP is 0.06 mM. Natural RNAs, poly(A) and poly(AG) are the best RNA primers among several homopolymers and copolymers tested. The poly(A) polymerase does not show any specificity for the nucleotide in the 3′ end of the RNA primer. The product of the reaction is a polyadenylic acid chain covalently bound to the RNA primer molecule. The length of the poly(A) chain is about ten nucleotides, but this length is dependent on the incubation time and the RNA primer concentration. The molecular weight of the enzyme is 70 000 and its isoelectric point is 6.0. The existence of an active poly(A) polymerase in dormant embryos of Artemia, likely with a cytoplasmic localization, suggests a role for this enzyme in the processing and activation of the stored mRNAs after resumption of development.

Early dolichyl sugar synthesis during differentiation of non-growing brine-shrimp (Artemia salina) embryos.

Incubation of cell-free extracts of Artemia salina with sugar nucleotides resulted in the formation of dolichyl derivatives. These enzymic activities were detected early in the development of the encysted Artemia embryos. Dolichyl sugars seemed to act as intermediates in the glycosylation of proteins, which may be involved in the initiation of cellular differentiation.

Protein Synthesis in Brine Shrimp Embryos

We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co-eIF-2(A) and Co-eIF-2(B). These factors are essential for ternary complex formation and binding of [35S]-Met-tRNAf to 40-S ribosomal subunits with 15-30 microgram eIF-2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co-eIF-2(A) is composed of a single polypeptide of Mr 65 000, whereas Co-eIF-2(B) contains polypeptides of Mr 105000 and 112000. Co-eIF-2(A) is sensitive to 4.5 microM aurintricarboxylic acid but Co-eIF-2(B) requires approximately 15 microM aurintricarboxylic acid to give 50% inhibition of ternary complex formation. The stimulatory activity of both factors is abolished by pretreatment of the proteins with N-ethylmaleimide. Artemia eIF-2 rapidly bonds [3H]GDP or [3H]GTP and at 15 degrees C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co-eIF-2(A) and Co-eIF-2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit (Mr 43 000) of Artemia eIF-2 is phosphorylated in the presence of the rabbit reticulocyte heme-repressible kinase. Tryptic digestion of [32P]phosphorylated eIF-2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF-2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF-2 is active in ternary complex formation, in AUG-dependent binding of [35S]Met-tRNAf to 40-S ribosomal subunits and in cell-free protein synthesis. Both Co-eIF-2(A) and Co-eIF-2(B) stimulate ternary complex formation with phosphorylated eIF-2. A kinase that phosphorylates the small subunit of eIF-2 is present in the post-ribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos.

Relations between latent membrane-bound and active polyribosomal messenger ribonucleoprotein particles in dormant and developing Artemia embryos.

Relations between latent membrane-bound and active polyribosomal messenger ribonucleoprotein particles in dormant and developing Artemia embryos.

The presence of guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine in brine shrimp embryos.

Acid-soluble extracts of dormant embryos of the brine shrimp, Artemia salina, contain small amounts of two previously undescribed dinucleotides which we have identified to be guanosine 5'-diphospho-5'-guanosine and guanosine 5'-triphospho-5'-adenosine. These compounds each comprise about 0.03% of the dry weight of the encysted embryos and are related chemically to guanosine 5'-triphospho-5'-guanosine and guanosine 5'-tetraphospho-5'-guanosine which have been shown previously to be major constituents of the nucleotide pool of Artemia cysts. These new dinucleotides were purified from perchloric acid extracts of dormant cysts by ion exchange column chromatography and identified by means of chemical, spectrophotometric, and enzymatic analyses compared to commercially available compounds. The possible role of these new compounds in nucleotide and nucleic acid metabolism in Artemia embryos is discussed.

DNA-dependent RNA-polymerases from Artemia embryos : Characterization of polymerases I and II from nauplius larvae

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.

Control of protein synthesis in brine shrimp embryos by repression of ribosomal activity

Undeveloped encysted embryos of the brine shrimp, Artemia salina, contain a large quantity of metabolically repressed 80S ribosomes. These ribosomes appear to be inactive or nonfunctional due to the presence of an inhibitor protein on their 60S subunit. During development the inhibitor is released or inactivated and the 80S ribosomes and their constituent subunits become fully functional in a poly(U)-directed protein-synthesizing system. The inefficiency of most 80S ribosomes from undeveloped Artemia embryos appears to be due to their inability to form stable complexes with poly(U) and phe-tRNA in the presence of elongation factor, EF-1. A potent inhibitor of protein synthesis has also been found in the 105,000 g supernatant fraction from undeveloped Artemia embryos. The exact nature of this inhibitor has not been ascertained but it appears to be a heat-labile protein devoid of RNase and protease activity. It is not known whether this inhibitor is the same as that associated with 60S ribosomal subunits of undeveloped cyst ribosomes.

Effect of anoxia on nucleotide metabolism in encysted embryos of the brine shrimp

When encysted embryos of the brine shrimp, Artemia salina , are maintained in an oxygen-free saline medium, morphogenesis and carbohydrate metabolism are arrested, but nucleotide metabolism continues in an orderly manner. Although anoxia inhibits the utilization of diguanosine 5′-tetraphosphate (Gp 4 G) in encysted embryos compared to embryos exposed to air only, Gp 4 G is metabolized under anaerobic conditions and appears to be the primary source of phosphate-bond energy necessary to maintain the viability of anoxia-arrested embryos. We found that during 4 months of anoxia approximately 50% of the total available phosphate-bond energy in the embryo, mainly in the form of Gp 4 G, is utilized. Also, during prolonged periods of anoxia the adenosine nucleotide pools are depleted, but guanosine nucleotide metabolism continues. When anoxia is terminated by aeration all nucleotide pools, except for the diguanosine nucleotides, return to levels found in control embryos at the time of hatching. In addition, the rate of recovery of the nucleotide pools correlates well with the delay, if any, in the resumption of morphogenesis. Finally, as development of Artemia embryos approaches hatching, N 2 treatment appears to have a diminishing effect upon the control of guanosine nucleotide metabolism.

Yolk platelets in brine shrimp embryos: Site of biosynthesis and storage of the diguanosine nucleotides

Yolk platelets have been isolated and purified from encysted embryos of the brine shrimp, Artemia salina, and found to contain 74.1% protein, 8.1% lipid, 4.0% carbohydrate, 4.6% acid-soluble nucleotides, and 0.1% DNA. The major component of the acid-soluble nucleotide fraction is diguanosine 5′-tetraphosphate (79%), and we have demonstrated unequivocally that the diguanosine nucleotides are localized in the yolk platelets of Artemia embryos and not in the nuclei as claimed by other investigators. In addition, we have presented evidence which indicates that the diguanosine nucleotide biosynthetic mechanism is also localized in the yolk platelets of Artemia embryos.

Nuclear localization of diguanosine polyphosphates in Artemia embryos

Diguanosine 5′-tetraphosphate and diguanosine 5′-triphosphate, previously known to occur in embryos of the brine shrimp Artemia salina, are present almost exclusively in the nucleus of Artemia cyst cells and, although decreasing in amount, remain in the nucleus during development of the cysts to swimming larvae. Their intranuclear localization raises the question of a possible regulatory role of these compounds during embryonic development of this organism.

Dissociation of ribosomes from dormant cysts of Artemia salina in potassium-free media

Ribosomes from dormant cysts of Artemia salina have a low endogenous activity of amino acid incorporation, but readily polymerize phenylalanine in the presence of polyU. Their activity of polyU-dependent phenylalanine incorporation is rapidly lost in K + or Mg 2+ deficient media. Increasing the MgCl 2 concentration from 1.5 mM to 5–10 mM does not effectively protect the ribosomes against inactiyation through K + deficiency. The endogenous amino acid incorporation by ribosomes from developing embryos is not markedly affected by K + deficiency under comparable conditions. The inactivation of the ribosomes from unincubated cysts in K + or Mg 2+ deficient media is correlated with a dissociation of the particles into subunits. The dissociation by K + deficiency is somewhat delayed, but not prevented by increasing the MgCl 2 concentration from 1.5 mM to 5–10 mM; 5–10 mM NH 4Cl is highly protective, but NaCl less effective. Subunits are formed in KCl-containining media already at moderately reduced MgCl 2 concentrations. However, their stability is less satisfactory, apparently due to K +-stimulated activation of latent ribosomal nucleases. The experiments indicate that the ribosomal monomers characteristic of early Artemia embryos are selectively inactivated and dissociated in K +-deficient media. Analogous conditions may occur in other cell materials with low endogenous ribosomal activity.

Dimethyl sulfoxide: failure to penetrate encysted Artemia embryos.

Dimethyl sulfoxide: failure to penetrate encysted Artemia embryos.