High-performance liquid chromatographic system for separating sugar phosphates and other intermediary metabolites.

A simple method for separating intermediates of carbohydrate metabolism, including the difficult-to-resolve sugar phosphates, using anion-exchange high-performance liquid chromatography is described. A gradient of decreasing borate concentration (10 to 0 mM) and increasing ionic strength (0 to 150 mM NH4Cl) separates phosphorylated sugars based on the strength of the ester complex that they form with borate anion. Lyophillized samples are reconstituted in a low ionic strength aqueous medium (5 mM triethanolamine-HCl, pH 8.1) and chromatographed on a commercially available anion-exchange column (Hamilton PRP-X100). The process requires only 3 h and permits nanomole detection of the phosphorylated intermediates of the glycolytic and pentose shunt pathways.

The in vivo rate of glucose-6-phosphate dehydrogenase activity in sea urchin eggs determined with a photolabile caged substrate.

Some of the earliest metabolic changes after fertilization of sea urchin eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of this shunt, as assayed with a photolabile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-3H or 1-14C) glucose and loaded into unfertilized sea urchin eggs by transient electroporation. Irradiation of these eggs (either before or after fertilization) photolyses the caged G6P, thereby pulsing the cell with 3H- and 14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to 3H2O and 14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1992) Exp. Cell Res. 201:366-372), the rate of 14CO2 production by the pentose shunt is equal to the flux of G6P through G6PDH. The data indicate that G6PDH activity is very low in unfertilized eggs, increases 184- to 427-fold by 2 min after fertilization, and then decreases to a value that is 74 to 209 times the unfertilized level (maximally 0.005 x 10(-8) units per egg in unfertilized eggs, 2.14 x 10(-8) units per egg by 2 min after fertilization, and 1.05 x 10(-8) units per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared with activity in broken cell extracts, G6PDH at these developmental times operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectively, of its potential activity. These results are discussed in terms of various hypotheses regarding the modulation of G6PDH activity by fertilization. These activity measurements relate well to other indices of in vivo activity. The major use of the NADPH shortly after fertilization is to produce H2O2, which is used as a substrate for fertilization membrane hardening; our data indicate that the NADPH that is produced by the pentose shunt activity is 30-70% of that required for this postfertilization generation of H2O2.

The use of caged substrates to assess the activity of 6-phosphogluconate dehydrogenase in living sea urchin eggs.

As part of our inquiries into the regulation of the hexose monophosphate shunt in the early development of sea urchin eggs and embryos, we have developed a novel method to assess the in vivo activity of the enzyme 6-phosphogluconate dehydrogenase (6PGDH) before and after fertilization. Our measurements show that the intracellular level of 6-phosphogluconate (6PG) in eggs decreases 60% after fertilization, which is consistent with the increase in the activity of 6PGDH previously reported using irreversibly permeabilized cell assays (Swezey and Epel, Proc. Natl. Acad. Sci USA 85, 812-816, 1988). The in vivo turnover of the 6PG pool was assessed using a new radioisotopic technique. 1-14C-labeled 6PG was chemically modified such that it was not metabolized by cellular 6PGDH and could be rapidly converted back to 6PG by photolysis. This "caged" 6PG was introduced into unfertilized sea urchin eggs using a transient permeabilization procedure, and then the oxidation of [1-14C]6PG in vivo upon irradiation was followed. Oxidation of 6PG was complete within 7-11 s of irradiation, indicating an extremely rapid turnover of this pool in sea urchin eggs. Based on the 6PG pool sizes and the kinetic properties of 6PGDH, determined here, along with the activity levels seen in permeabilized cells, the half-time for the label in the 6PG pool in sea urchin eggs is calculated to be 26 s. This is inconsistent with the in vivo turnover rates seen in these studies, indicating that the permeabilized cell assays overestimate the degree of inhibition of 6PGDH before fertilization. These results suggest that caution should be exercised in extrapolating data obtained from permeabilized cells to the situation in vivo.

Stable, resealable pores formed in sea urchin eggs by electric discharge (electroporation) permit substrate loading for assay of enzymes in vivo.

We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.

Enzyme stimulation upon fertilization is revealed in electrically permeabilized sea urchin eggs.

Sea urchin eggs and embryos subjected to high-voltage electric discharge in a medium mimicking the intracellular milieu retain their structural integrity and remain permeable, permitting substrates to enter the cytoplasm and thus assay of enzyme activity. At saturating concentrations of substrates, five of six enzymes assayed for more active (three to fifteen times) in permeabilized embryos than in permeabilized eggs, but no fertilization-related differences are seen in homogenates prepared from these same permeabilized cells. Furthermore, enzyme activity in homogenates always exceeds that in the permeabilized cell suspensions. This difference in enzyme reaction rates between unfertilized eggs and fertilized eggs is not due to differences in the diffusibility of substrates into the permeabilized cells. The activity of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) in permeabilized cells was studied in greater detail and has the following characteristics. (i) Regulation of activity persists during early development. (ii) This regulation is not mediated by diffusible allosteric agents. (iii) Stimulation at fertilization is initiated by a rise in intracellular calcium and is further promoted by cytoplasmic alkalinization. (iv) The microenvironment experienced by this enzyme intracellularly differs from that of the enzyme in homogenates as evidenced by markedly different pH vs. activity profiles. These results indicate that the regulatory status of enzymes is preserved in electrically permeabilized cells and suggest that this regulation depends on some cell structural feature(s) that is (are) destroyed upon homogenization.

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs.

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.

Regulation of glucose-6-phosphate dehydrogenase activity in sea urchin eggs by reversible association with cell structural elements.

In unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, glucose-6-phosphate dehydrogenase (G6PDH) associates with the particulate elements remaining either after homogenization or extraction of eggs with non-ionic detergent in low ionic-strength media. At physiological ionic strength, the extent of G6PDH binding to these particulate elements is proportional to the total protein concentration in the extracts. In fertilized eggs this association is prevented by one or more low molecular weight solutes. The dissociation is reversible, and there are no permanent modifications of either G6PDH or its particulate binding site that affect binding. After fertilization, the time course of dissociation of G6PDH from particulate elements is too fast to be caused by a change in intracellular pH, but it could be triggered, but not maintained, by an increase in the intracellular calcium concentration. Binding of G6PDH to the particulate fraction lowers its catalytic activity at all substrate concentrations. Therefore, release of the enzyme into the cytoplasm may be an important part of the suite of events causing metabolic activation of the egg at fertilization.

Pressure effects on actin self-assembly: interspecific differences in the equilibrium and kinetics of the G to F transformation.

Purified skeletal muscle actins from species whose ambient pressures range from 1 to greater than 500 atm were examined for the sensitivity to hydrostatic pressure of the globular (G) to filamentous (F) self-assembly reaction. Both the equilibrium position and the kinetics of self-assembly were affected by pressure. Increased pressure shifted the self-assembly equilibrium toward the monomer (G) state and reduced the rate of F-actin assembly. For most of the actins studied, the perturbation by pressure of F-actin formation decreased with increasing measurement of pressure, indicating that F-actin has a higher compressibility than G-actin. The increase in system volume and compressibility concomitant with the assembly of F-actin can be interpreted as reflections of the major role played by hydrophobic effects in stabilizing F-actin and of the existence of "hard" binding sites, in the terminology of Torgerson et al. [Torgerson, P. M., Drickamer, H. G., & Weber, G. (1979) Biochemistry 18, 3079-3083], in the actin subunits. For actin from the deepest occurring species studied, the teleost fish Coryphaenoides armatus, which occurs to depths of approximately 5000 m (equivalent to 501 atm of pressure), there was no difference in compressibility between G-actin and F-actin; that is, the effect of increasing pressure on self-assembly was linear over the entire pressure range examined, 600 atm. The self-assembly reaction of the actin from C. armatus also differed from that of the other actins examined in that the G to F equilibrium was relatively insensitive to increased pressure; i.e., the volume change (delta V) of assembly was small.(ABSTRACT TRUNCATED AT 250 WORDS)