Ingrid M. Petersen

Natural occurrence, and experimental induction by estradiol-17-β, of a lipophosphoprotein (vitellogenin) in flounder (Platichtys flesus, L.)

Abstract 1. 1. A specific lipophosphoprotein, vitellogenin is demonstrated in female flounders during vitellogenesis. 2. 2. The synthesis of vitellogenin can be induced in both normal males and females by estradiol-17-β treatment. In vitellogenic females the estradiol-treatment increases the synthesis of vitellogenin. 3. 3. Determination of alkali-labile protein phosphorus is shown to be a useful indication of vitellogenin in serum. 4. 4. By subjecting serum of vitellogenic animals to gel filtration it is possible to isolate a crude preparation of vitellogenin, which has a molecular weight of about 550,000.

Vitellogenin, lipid and carbohydrate metabolism during vitellogenesis and pregnancy, and after hormonal induction in the blenny Zoarces viviparus (L.).

1. Ovarian vitellogenic growth in Zoarces viviparus lasts about 2 months. Vitellogenesis is immediately followed by ovulation, fertilization and a pregnancy period of 4 months. Vitellogenin is observed in the blood during vitellogenesis, but declines during the first month of pregnancy. 2. The largest amount of liver lipid is found before vitellogenesis is initiated. During pregnancy the liver is depleted of lipid and glycogen, and total lipid and phospholipid is accumulating in the blood. 3. Estradiol treatment during pregnancy results in a dose-dependent increase in vitellogenin and lipids of the blood. 4. In late pregnancy, birth can be provoked with progesterone alone, or with combined progesterone and estradiol treatment.

Changes in serum glucose and lipids, and liver glycogen and phosphorylase during vitellogenesis in nature in the flounder (Platichtys flesus, L.).

1. During vitellogenesis in nature the concentration of phospholipid in serum of female flounders is correlated to the concentration of vitellogenin. 2. High levels of glucose and total lipid in blood occur before the onset of vitellogenesis. During early vitellogenesis the values decrease. At spawning they reach maximum levels. After spawning they decrease to low levels. 3. The concentration of glycogen in the liver oscillates during the period August to May. The phosphorylase activity shows peak activities with high concentrations of glycogen in the liver. There is no simple correlation between glycogen concentration in the liver and glucose concentration in the blood.

Changes in some carbohydrate metabolizing enzymes and glycogen in liver, glucose and lipid in serum during vitellogenesis and after induction by estradiol-17-β in the flounder (Platichtys flesus L.)

Abstract 1. 1. Parallel with the increase in the concentration of vitellogenin in serum in nature, (October-November) the activity of the gluconeogenetic enzymes FDP-ase and G-6-P-ase decreases and the glycolytic enzymes PK and PFK increases. 2. 2. In the spawning period in January to May the activity of PFK and G-6-PDH decreases, and the activity of FDP-ase increases. 3. 3. Estradiol-treatment of the males levels off the sex-bound differences in the measured carbohydrate metabolizing enzymes compared to the natural vitellogenic female and increases the activity of the carbohydrate metabolizing enzymes in both sexes. 4. 4. Estradiol-treatment increases the serum concentrations of vitellogen and phospholipid in both sexes and decreases the glucose concentration in the male flounder.

Dose response kinetics of serum vitellogenin, liver DNA, RNA, protein and lipid after induction by estradiol-17β in male flounders (Platichthys flesus L.)

1. Male flounders receiving 100 micrograms estradiol each second day were fully induced to vitellogenin synthesis within 11 days, while fishes given 5 micrograms doses continued to accumulate vitellogenin in the serum at a progressive rate through 17 days. 2. Liver DNA per unit fish remained constant, while RNA per unit fish in flounders given 100 and 5 micrograms doses attained values 80 and 25% respectively, above the values found in control animals. 3. Liver RNA per unit DNA increased at maximal rate within 6 days in fishes receiving 100 micrograms doses. RNA synthesis continued at a progressive rate through 17 days in fishes given 5 micrograms doses of estradiol. 4. Liver protein per unit DNA elevated at a plateau 60% above control within 6 days with 100 micrograms doses. Doses of 5 micrograms had only little effect on liver protein. 5. Estradiol had a lipogenic effect on the liver. Cellular lipid rose 120 and 60% above control after treatment with 100 and 5 micrograms respectively. 6. Liver dry weight per unit DNA increased 60 and 55% above control with 100 and 5 micrograms doses respectively. Cellular hypertrophy in fishes receiving the smaller dose was primarily associated with an increase in lipid concentration, while protein and lipid contributed almost equally to cellular growth in fishes receiving the high dose.

Activities of carbohydrate and amino acid metabolizing enzymes from liver of mink (Mustela vison) and preliminary observations on steady state kinetics of the enzymes

The activity and some kinetic parameters of the key enzymes of the glycolysis, the gluconeogenesis and the amino acid catabolism from the liver of male and female mink have been determined and compared to the corresponding activities from rat and cat. The activities of glucose-6-phosphatase and pyruvate kinase are dependent on sex, both being higher in females. Except for pyruvate carboxylase the glycolytic and the gluconeogenic enzyme activities of the mink are higher than those of rat and cat; especially the activities of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are markedly higher. The activities of glutamate dehydrogenase and glutamate oxaloacetate transaminase are smaller than the corresponding activities of rat but higher than those of cat. The results suggest that mink has a high capacity for gluconeogenesis compared to rat.

A time course study of the effect of repetitive doses of estradiol-17β on serum glucose and lipids, liver glycogen and some carbohydrate metabolizing enzymes in liver of male flounder (Platichtys flesus L.)

1. Repeated treatment of male flounder with 5 and 100 microgram doses of estradiol-17 beta increases the level of phospholipid, triglyceride, free fatty acids and total lipid in serum as a function of time and dose, during a period of 17 days. 2. The glucose level in serum is increased and decreased respectively by doses of 5 and 100 micrograms. 3. Five and 100 microgram doses decrease the level of glycogen in liver. 4. Five microgram doses do not affect the activity of the measured enzymes considerably, with the exception of phosphorylase a. 5. One hundred microgram doses increase the activity of glucose-6-phosphate dehydrogenase after 11 days. 6. One hundred microgram doses increase the activity of pyruvate kinase continuously during the experimental period and decrease phosphorylase a and glucose-6-phosphatase activity.

Estradiol-induced hepatic protein synthesis and transaminase activity in the male flounder, Platichthys flesus (L.)

A continued accumulation of vitellogenin was observed in the serum through 28 days after administration of spaced large doses of estradiol. The level of estradiol in the serum reached a maximum on Day 4, followed by a continued decrease until a basal concentration was reached at Day 15. The estradiol treatment increased the in vitro hepatic protein synthesis activity, measured as polyphenylalanine synthesis per unit cytoplasmic RNA, within 2-4 days reaching a maximum after 7 days. The effect was transitory and reduced to less than 50% within 15 days. After restimulation a second rise in protein synthesis activity was observed. The cellular content of bulk RNA i the liver showed a slow but steady accumulation through the 28 days. The specific activity of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase in the liver decreased 35 and 20% relative to control, respectively, within 7 days after the first stimulation and again 14 days after restimulation. The concentration of serum protein showed a steady increase throughout the experimental period. The concentration of serum ninhydrine positive substances was lowered to 50% relative to the controls within the first 7 days after start of treatment with hormone.

Experimental induction of vitellogenin synthesis in eel (Anguilla anguilla) adapted to sea-water or freshwater

Abstract 1. 1. One week after treatment with a single dose of estradiol, the vitellogenin (Vg) concentration was significantly higher in the freshwater group than in the sea-water group. 2. 2. DNA concentration in the liver was increased in the freshwater group compared to the control, while there was no effect on the sea-water group. 3. 3. RNA concentration in the liver was significantly increased in both groups compared to the controls. 4. 4. Treatment with estradiol did not affect the osmotic concentration when compared to the control group, but a significant decrease is seen in the freshwater control group compared to the sea-water control group. 5. 5. Treatment with estradiol decreased the concentration of chloride in plasma in both sea-water and freshwater groups compared to the controls. 6. 6. Gel filtration of plasma from estradiol-treated animals shows that the elution profile contains a peak, which coincides with a Vg peak. 7. 7. One week after treatment with estradiol the concentration of estradiol in plasma in the treated sea-water group was twice as high as in the treated freshwater group.

Purification and some properties of glyceraldehyde 3-phosphate dehydrogenase from Synechococcus sp.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was purified 386 fold to apparent homogeneity from the thermophilic cyanobacteriumSynechococcus sp. grown at optimum light intensities in batch cultures. The molecular mass of the tetrameric form of the enzyme was 160 kDa as determined by gel filtration and sucrose gradient centrifugation in a phosphate buffer containing DTT. The pH optimum for the oxidation of NADPH was broad (6–8) and the enzyme had a pI of 4.5. The turnover number was 36,000 min−1 at 40° C. The activation energy was 12.4 Kcal for t>29° C and 20.6 Kcal for t<29° C. The specific absorption coefficient, A280 mm1% 1cm of the pure enzyme in phosphate buffer at pH 6.8 was 15.2.By SDS gel electrophoresis molecular masses of 78 kDa and 39 kDa were found, indicating that the purified enzyme is a tetramer, probably a homotetramer.When Tris was used as buffer in the homogenization and phosphate and DTT were omitted, a high molecular form with a molecular mass above 500 kDa was found. This form was less active than the purified tetrameric form. Acetone and other organic solvents stimulated the native enzyme several fold.