Partial purification and properties of the reticulocyte guanylating enzyme.

The guanylating enzyme which catalyzes the insertion of a guanine residue into one of the isoacceping tRNAHis of rabbit reticulocytes has been purified approximately one-hundred fold. It is free of nuclease activity. The enzyme does not catalyze the replacement of inserted radioactive guanine by unlabeled guanine, indicating that the reaction is irreversible. We have separated the histidyl-tRNA of reticulocytes into three isoacceptors. Previous work showed that the last histidyl-tRNA to elute from RPC-5 columns was the product of the guanylation reaction. This reports shows that the same late-eluting peak also contains the substrate for the guanylating enzyme, indicating that the guanine insertion reaction is chromatographically silent. The isoaccepting tRNAHis that is the substrate for the guanylating enzyme does not contain the hypermodified base known as Q. It is the other major reticulocyte tRNAHis that coantains Q, showing that at least in the reticulocyte the role of the guanylating enzyme is not the conversion of the Q form of tRNA to the homogeneic G form. The purified enzyme does not insert any base other than guanine into tRNA.

In vitro synthesis and processing of components of the Ascaris suum pyruvate dehydrogenase complex.

Poly(A)+ RNA was isolated from Ascaris suum body wall muscle and translated in a cell-free rabbit reticulocyte lysate system. Specific antisera and immunoglobulins against the alpha-pyruvate dehydrogenase and dihydrolipoyl transacetylase components of ascarid pyruvate dehydrogenase complex were used to immunoprecipitate individual radiolabelled polypeptides from the in vitro translation mixtures. Both polypeptides appeared to be synthesized as preproteins about 1.5 and 8 kDa larger than the corresponding native proteins. Incubation of the dihydrolipoyl transacetylase preprotein with an ascarid high-speed mitochondrial supernatant fraction resulted in the formation of a polypeptide with apparent molecular weight intermediate in size between the preprotein and the native enzyme. This processing was insensitive to phenylmethylsulfonyl fluoride and leupeptin but was completely abolished by EDTA. These results suggest that in A.suum, as in other organisms, mitochondrial matrix proteins coded by the nuclear genome are synthesized as larger preproteins and processed by a specific, metal-dependent mitochondrial matrix protease.

Immunochemical characterization of the pyruvate dehydrogenase complex in adult Ascaris suum and its developing larvae.

Polyclonal antibody was prepared against the pyruvate dehydrogenase complex purified from adult Ascaris suum body wall muscle. The antibody reacted with the E2, X, alpha E1 and beta E1 subunits of the complex in immunoblots of mitochondrial supernatant fractions and homogenates of adult muscle. In addition, the same subunits were observed in immunoblots of homogenates of L3 and L4 ascarid larvae, suggesting that a similar enzyme complex was present in all developmental stages despite their marked differences in energy metabolism. The phosphorylated and dephosphorylated alpha E1 peptides migrated differently during sodium dodecylsulfate polyacrylamide gel electrophoresis and both forms of the enzyme were recognized by the antibody. These results and those obtained with ELISA suggest that both phosphorylated and dephosphorylated forms of the alpha E1 subunit react equally well with the antibody. In immunoblots of adult body wall muscle, the phosphorylated alpha E1 peptide predominated, while immunoblots of L3 larvae contained predominantly the dephosphorylated form. These results reflect the in vivo activity state of the pyruvate dehydrogenase complex in these two stages and suggest that this technique may be useful for determining the activity state of enzyme complex directly from immunoblots of homogenates A. suum and other helminths.

Estradiol 17 beta decreases the in vitro phosphorylation of a 49K cytosolic protein from the adult female rat preoptic-hypothalamus.

Acute treatment (2 hours) with estradiol resulted in decreased in vitro phosphorylation of a specific 49,000 MW protein in cytosolic extracts of female preoptic-hypothalamus from intact and ovariectomized rats. Estradiol had no effects on the phosphorylation of this or any other cytosolic protein from the estradiol-insensitive female cortex.

The effect of neonatal testosterone on specific male and female patterns of phosphorylated cytosolic proteins in the rat preoptic-hypothalamus, cortex and amygdala.

This study demonstrates that in the rat there are specific patterns of in vitro phosphorylation of cytosolic proteins for the preoptic-hypothalamus, cortex, and amygdala. Furthermore, there are sex-specific patterns for the male and female preoptic-hypothalamus. These sex-specific patterns are controlled by the sex steroid environment of the neonatal rat. If testosterone was removed by neonatal castration of a male, the female pattern of in vitro phosphorylated proteins was found in the adult preoptic-hypothalamus. Conversely, if a neonatal female was androgenized at 2 days by a single injection of testosterone, a male-like pattern was found in the adult preoptic-hypothalamus. Treatment of neonatal females or males did not alter the adult patterns of in vitro protein phosphorylation in the cortex, but such treatments did give rise to anomalous patterns in the amygdala. Thus, as with behavioral and structural characteristics of the preoptic-hypothalamus, the spectrum of phosphorylated proteins in this region also seems to be organized by neonatal testosterone.