Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes.

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.

Estrogen action in rooster liver. Modification of a tRNASer-inactivating activity.

The hepatic synthesis of the yolk protein, phosvitin, can be induced in rooster by the administration of estrogen and this provides a model for studying the regulatory mechanisms involved. Since phosvitin is unusually rich in serine the effect of estrogen on the metabolism of serine transfer RNA (tRNASer) was examined. Rooster liver contains an activity capable of inactivating rooster liver tRNASer. This activity has specificity in that it inactivates only part of the tRNASer in the unaminoacylated form. Upon estrogen administration this activity is modified with a change in specificity and the modified activity is able to inactivate only a significantly smaller amount of tRNASer than the unmodified activity. This novel mechanism may be involved in the regulation of some species of tRNASer and thus may be part of the translational control involved in estrogen-induced phosvitin synthesis.

Phosvitins in Fundulus oocytes and eggs. Preliminary chromatographic and electrophoretic analyses together with biological considerations.

Vitellogenin serves as the plasma precursor for the yolk proteins, lipovitellin and phosvitin, in nonmammalian vertebrates. 32P-Vitellogenin was isolated from the plasma of the teleost, Fundulus heteroclitus, and was used both to label phosvitin in the ovary and to indicate the phosvitin region in preparative chromatographs of ovarian extracts on DEAE-cellulose. Crude [32P]phosvitin could be resolved further into two labeled components with shallow gradients on DEAE-cellulose and into eight labeled components by electrophoresis on 12% polyacrylamide gels. Only the two largest electrophoretically resolved components could be correlated with Coomassie Blue staining bands, but several of the smaller components could be indicated with the cationic carbocyanine dye, Stains-all. Stains-all-dyed components were also generally indicated as multiple bands. The ovary of a reproductively active female contains vitellogenic oocytes, postvitellogenic oocytes undergoing maturation prior to ovulation, and ovulated eggs. Examination of various types of follicles and eggs on polyacrylamide gels revealed that during maturation, the largest phosvitin components formed during vitellogenesis either disappear or diminish, while smaller phosvitin components appear. The transformation of phosvitin components can also be achieved in vitro by incubating prematurational follicles in a saline medium containing deoxycorticosterone. These preliminary results demonstrate that a complex array of phosvitin-like components are present within a single ovary of F. heteroclitus. We also postulate that one reason for the anomalous yolk proteins generally found thus far in teleost eggs is that some of the proteins derived from vitellogenin during vitellogenesis undergo further proteolysis during oocyte maturation.

Comparative study of hen yolk phosvitin and plasma vitellogenin.

Vitellogenin, the only phosphoprotein detectable in the plasma of laying hens, is present at an approximate concentration of 1 mg/mL and can be isolated by chromatography on diethylaminoethylcellulose. Vitellogenin has a molecular weight of 235 000--240 000 and contains approximately 3% phosphorus by weight. Evidence that this protein is the precursor of phosvitins includes its ability to act as an acceptor for phosphate with a phosvitin specific kinase, the generation of a peptide similar to phosvitin by trypsinization, and the presence of distinctive peptides of multiple clustered phosphoserine upon partial acid hydrolysis. This partial sequence similarity between phosvitins and vitellogenin has not been previously reported. The phosphorus content and amino acid composition of vitellogenin are consistent with a model which contains two phosvitins and one lipovitellin. The total molecular weights of these proteins (28 000 + 34 000 + 170 000 = 232 000) are close to that of vitellogenin.

Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells.

Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both 3H-uridine uptake and incorporation into total RNA, a decline of 3H-leucine and 14C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.

Studies on the mechanism of in vitro estradiol-17 beta induced synthesis of phosvitin in chick embryo liver cells.

The effect of estradiol-17-beta treatment on phosvitin synthesis by cultured chick embryo liver cells has been studied. Phosvitin synthesis occurs approximately 15 hr of hormone treatment; the synthesis being blocked by actinomycin D treatment suggests that RNA synthesis is required. The life time of the newly synthesized RNA is at least 24 hr. The significance of these findings with respect to the mechanisms involved in hormone-mediated protein synthesis is discussed.

Precursor-product relationship between vitellogenin and the yolk proteins as derived from the complete sequence of a Xenopus vitellogenin gene.

In Xenopus laevis four estrogen-responsive genes are expressed simultaneously to produce vitellogenin, the precursor of the yolk proteins. One of these four genes, the gene A2, was sequenced completely, as well as cDNAs representing 75% of the coding region of the gene. From this data the exon-intron structure of the gene was established, revealing 35 exons that give a transcript of 5,619 bp without the poly A-tail. This A2 transcript encodes a vitellogenin of 1,807 amino acids, whose structure is discussed with respect to its function. At the nucleic acid as well as at the protein level no extensive homologies with any sequences other than vitellogenin were observed. Comparison of the amino acid sequence of the vitellogenin A2 molecule with biochemical data obtained from the different yolk proteins allowed us to localize the cleavage products on the vitellogenin precursor as follows: NH2 - lipovitellin I - phosvitin (or phosvette II - phosvette I) - lipovitellin II - COOH.

Subcellular redistribution of seryl-transfer RNA during estrogen-induced phosvitin synthesis and specificity of the estrogen effect.

The estrogen-induced hepatic synthesis of the serine-rich protein, phosvitin, in chickens is accompanied by an increase in the serine acceptance of total hepatic tRNA, which is limited to two sering isoacceptors. To investigate the role of the tRNA alterations in the synthesis of phosvitin, the relative amounts of various seryl-tRNA species in isolated nuclei and in free and membrane-bound ribosomes were determined. Estrogen treatment causes a shift in the subcellular distribution of hepatic seryl-tRNA. Of the four serine isoacceptors, the amount of tRNASerAGU,AGC was specifically increased in nuclei and in membrane-bound ribosomes. Changes in total hepatic tRNA occurring during physiologic estrogenization were compared with those occurring by varying steroid hormones, times after estrogen administration, estrogen doses, animal ages, and tissue types. The changes observed demonstrate that the seryl-tRNA alterations are closely correlated with the synthesis of phosvitin. The coincident change in seryl-tRNA levels and in phosvitin synthesis, together with the specific change in cellular localization, suggests that the amount and subcellular distribution of each tRNA species are separately controlled in a manner dependent upon its frequency of use in translation.