cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

Effect of reduced protein synthesis on the cell cycle in sea urchin embryos

We have reinvestigated the existence of cyclical fluctuations of protein synthesis and have examined the effects of reducing it in early embryos of the purple sea urchin, Strongylocentrotus purpuratus. The results show that protein synthesis increases linearly during the first 45-60 minutes after fertilization, then transiently decreases during mitosis, and rises again at first cleavage. Reducing protein synthesis of embryos to 35% its normal value only slightly affects the rate of progression through the cell cycle. It is also shown that the observed retardations of the cell cycle, under depressed protein synthesis, are attributable (by 80%) to a lengthening of the premitotic phase but also, to a lesser extent (20%), to a lengthening of the mitotic phase itself. These results suggest that mitotic proteins, in sea urchin embryos, are stable and little affected by an imposed decrease of protein synthesis during their accumulation phase. This analysis supports the view that specific mechanisms, other than decreased protein synthesis, need be turned on only at appropriate times during the cell cycle in order to explain the destruction or deactivation of mitotic proteins. Finally, a one-dimensional SDS-PAGE analysis of synthesized proteins, labeled with 35S-methionine, reveals the presence of a 50-kDa cyclin showing the expected characteristics of mitotic proteins deduced from our results.     

The synthesis of ribonucleic acid in sea urchin embryos

The sea urchin egg rapidly acquires the ability to synthesize protein after fertilization. This activation is due to a changing property of the ribosomes (1). Previous work has shown unfertilized egg ribosomes are capable of directed protein synthesis in the presence of polyuridylic acid (2, 3). The purpose of this report is to present evidence suggesting the hypothesis that the activation of ribosomes is a consequence of the activation of messenger RNA synthesis. Experiments will be described showing that the sedimentation properties of ribosomes from fertilized and unfertilized eggs are virtually identical. RNA synthesis is activated following fertilization, and a portion of this RNA is found in the ribosome fraction. The bulk of the newly synthesized RNA does not possess sedimentation properties of ribosomal RNA.     

Interaction between glutathione and the endoplasmic reticulum in cyclic protein synthesis in sea urchin embryos.

Interaction between an oxidoreduction system and cyclic protein synthesis was studied in sea urchin embryos. When assayed enzymatically, in both in vivo and in vitro systems, the contents of GSH and GSSG varied inversely in a cyclic fashion. Diamide at 0.5 mM inhibited amino acid incorporation in not only the cyclic phase but also the basal phase, but 4-nitroquinoline-N-oxide at 1 μM inhibited only the cyclic phase. Sea urchin embryos contained membrane-bound ribosomes, and pulse-labeling with amino acids suggested that free ribosomes were responsible for the basal phase and membrane-bound ribosomes were responsible for the cyclic phase of amino acid incorporation. Thiol-disulfide interchanging enzyme was found in the endoplasmic reticulum fraction. An extract of the endoplasmic reticulm caused stimulation of binding of acetylphenylalanyl-tRNA to 40S ribosomes and polyphenylalanine synthesis in the presence of low GSH concentrations. An extract of the endoplasmic reticulum also catalyzed oxidoreduction from GSH to the KCl-soluble protein. Thus, the periodic stimulation of protein synthesis is interpreted to be the result of the periodic activation of membrane-bound ribosomes by the thiol-disulfide interchanging enzyme which accepts selectively the signal from the GSH cycle.     

Nucleic acid and histone synthesis by ethanol-treated cleavage-arrested sea urchin embryos

It has been found that fertilized sea urchin eggs prevented from normal cleavage by solutions of isosmotic ethanol in sea water are able to complete some cellular and molecular aspects of the normal developmental program that are observed in control cultures. In both treated and control cultures, the type of RNA transcribed changes at 24 h (early gastrula) in favor of higher molecular weight rRNA. Ultrastructural studies reveal the presence of nucleoli in ethanol-treated as well as control embryos. The type of H1 histone synthesized also shifts at 24 h in favor of a higher molecular weight H1 in both ethanol-treated and control embryos. Replication of DNA proceeds at a slower rate in ethanol-treated embryos than in controls, resulting in DNA/embryo values in ethanol which are 20-30% of control values after 24 h. The results relate to the problem of differentiation without cleavage, and the role of normal partitioning, cell-cell interaction, and DNA synthesis in triggering the sequence of events in the developmental program.     

Terminal completion of poly(A) synthesis in sea urchin embryos.

The kinetics of accumulation of nuclear and cytoplasmic poly(A) have been determined in sea urchin blastulas and gastrulas, stages when essentially all mRNA is synthesized de novo in the nucleus. A majority of the labeled poly(A) is found in the cytoplasmic fraction after a brief pulse. The ratio of radioactive AMP to adenosine in pulse-labeled nuclear, cytoplasmic, and polyribosomal poly(A) is considerably less than the number average length of the labeled poly(A), indicating that there is 3′-terminal addition of adenosine to previously synthesized poly(A). The size distribution of pulse-labeled, terminally elongated poly(A) in the cytoplasm is similar to that of the largest nuclear poly(A) rather than the steady-state size distribution of cytoplasmic poly(A), which is smaller and more heterogeneous. The most likely interpretation of these results is that there is a predominant 3′ terminal addition of short tracts of adenosine to poly(A) attached to nuclear RNA just before or during entrance of this RNA into the cytoplasm. In this respect, much of the 3′ terminal addition may be thought of as terminal completion of poly(A) synthesis.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.     

Ciliary dynein from sea urchin embryos.

Axonemal dynein ATPase [EC 3.6.1.3] was extracted from cilia of sea urchin embryos for a study of its enzymatic properties. Sedimentation analysis on a sucrose density gradient revealed that ATPase activity was associated with the 12S particles. The partially purified 12S enzyme was characterized mainly with regard to the optimum pH, divalent cation and ionic strength requirments and substrate specificity. Comparative investigation of the data obtained indicates that the properties of the present dyneine ATPase resemble those of other dynein(-like) ATPase hitherto reported. In addition, the possible relationship among dyneins within a single species, in particular between the ciliary dynein and cytoplasmic dynein-like ATPase, is discussed.     

Tubulin synthesis in sea urchin embryos: Almost all tubulin of the first cleavage mitotic apparatus derives from the unfertilized egg

Developing embryos of Stronglyocentrotus purpuratus were exposed to [¹⁴C]leucine between fertilization and metaphase of the first cleavage division. At metaphase, mitotic apparatus was isolated from the embryos and tubulin was extracted from mitotic apparatus. The specific activity of the tubulin fraction was only 0.2 to 0.4 times the specific activity of whole embryo protein. We calculate from this result that no more than 0.4% of the tubulin of the first cleavage mitotic apparatus could be synthesized following fertilization.     

Stress proteins by zinc ions in sea urchin embryos

In Paracentrotus lividus embryos, treatment with zinc ions induces the synthesis of the two major stress proteins with the same molecular weight as those induced by heat shock. The developmental stages responsive to zinc ion treatment are the same as those responsive to heat shock. However, zinc treatment induces a longer lasting synthesis of the stress proteins, and, unlike heat shock, does not induce thermotolerance and does not inhibit synthesis of the bulk proteins.