Biomedical subjects

J L Fando

Publications and source records attributed to J L Fando.

35 records · Page 2Linked to original sources

PubMed1986

Protein kinase activities associated with ribosomes of developing rat brain. Identification of eukaryotic initiation factor 2 kinases.

Protein kinases associated with ribosomes in the brains of suckling (4-10 days) and adult (2 months) rats were extracted from ribosomal fraction with 0.5 M KCl. The different protein kinase activities were characterized by their ability to phosphorylate three exogenous substrates: casein, histone IIs and histone IIIs in the presence of different modulators. Ribosomal salt wash fractions contain a high casein kinase activity which was partially inhibited by heparin and stimulated by calmodulin in the presence of Ca2+, indicating the presence of casein kinase I and II and calcium/calmodulin-dependent kinases. Cyclic AMP and cyclic GMP-dependent kinases and protein kinase C (calcium/phospholipids-dependent kinase) were also present. No differences were found in the casein kinase activities of suckling and adult animals, but histone kinase activities were higher in adult than in suckling animals. To identify initiation factor 2 kinases, purified factor from adult brains was used as a protein marker. In addition to the phosphorylation of both factor subunits alpha and beta by casein kinase I or II, an increased phosphorylation was detected of alpha subunit in the presence of cyclic AMP, and beta subunit, in the presence of Ca2+/calmodulin or Ca2+/phospholipids. Present results reinforce our hypothesis that, as occurs in other eukaryotic cells, the decreased rate of protein synthesis during brain development may be regulated by phosphorylation of initiation factor 2.

Aging↗

PubMed1985

Initiation factor 2 isolated from rat brain contains kinase activities responsible for its phosphorylation.

Initiation factor 2 from adult rat brain was isolated from salt-washed microsomes using a three-step purification process consisting of heparin-Sepharose, phosphocellulose and diethylaminoethyl-cellulose (DEAE cellulose) column chromatographies. The initiation factor 2(eIF-2) was phosphorylated in subunits alpha and beta by the endogenous protein kinase activity present in the pruified preparation. This protein kinase activity proved to be mostly a casein kinase, although the possible presence of a very specific alpha kinase activity cannot be dismissed.

Animals↗

PubMed1985

Functional heterogeneity of GEF-free initiation factor 2 purified from suckling and adult rat brain.

The functional behavior of initiation factor 2 was studied in purified preparations from the brains of suckling (4-12-day-old) and adult (60-day-old) rats. Adult eIF2 has lower GDP and GTP affinity than suckling eIF2, even in the presence of a large excess of GTP, whereas suckling eIF2 has a lower capacity to bind GTP. Since these two factors are free of guanine nucleotide exchange factor (GEF), and ribosomal fractions show an age-dependent difference in GEF activity, the observed functional heterogeneity may be due to a different ratio in eIF2 species (eIF2-GDP, eIF2(alpha P)).

Aging↗

PubMed1985

The mode of action of restrictocin and mitogillin on eukaryotic ribosomes. Inhibition of brain protein synthesis, cleavage and sequence of the ribosomal RNA fragment.

The relationship between the process of rat brain protein synthesis inhibition by restrictocin and mitogillin and the induction of a specific cleavage in 28S rRNA has been examined. Restrictocin or mitogillin at a concentration of 6 nM inhibits protein synthesis to a level of 80%. The inhibition induced by 6 nM alpha-sarcin is of the same order, indicating that those molecules all catalytically inhibit protein synthesis. When restrictocin or mitogillin was reduced and alkylated, a 100-fold higher concentration of these chemically modified molecules was needed in order to obtain the same inhibition as with native molecules, suggesting a close correlation between activity and configuration of the inhibitors. The inhibitory activity of restrictocin or mitogillin was completely lost after oxidation with performic acid. The inhibition of protein synthesis was always correlated with the production of a fragment from the 3'-end of the 28S rRNA. This rRNA fragment had the same electrophoretic mobility as that produced by alpha-sarcin. The 5'-end sequence of the rRNA fragment produced by restrictocin, mitogillin or alpha-sarcin is AGGAA, demonstrating that restrictocin and mitogillin inhibit protein synthesis in the same way as does alpha-sarcin. The conservation of this region of the rRNA from archaebacteria to eukaryotic ribosomes indicates its importance in the elongation process.

Alkylation↗

PubMed1985

Isolation of eukaryotic initiation factor 2 from rat brain.

Eukaryotic initiation factor 2 (eIF-2) was isolated from salt-washed microsomes of 4-day-old rat brain which show a high rate of protein synthesis. A three-step purification scheme was employed, including heparin-Sepharose, phosphocellulose, and DEAE-cellulose column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated factor revealed three polypeptides with molecular weights of 43,000, 54,000, and 59,000 and 90% purity. The rat brain eIF-2 forms ternary complexes with [3H]methionyl-tRNAi and GTP. In terms of specific activity, the purification does not correspond to that revealed by electrophoretic analysis. During purification there is an apparent loss of additional factors that modulates the activity of eIF-2 and explains the high rate of activity of the crude fraction.

Animals↗

PubMed1985

The effect of streptozotocin diabetes on brain protein synthesis in the rat.

Young normal control rats were compared with a group made diabetic by treatment with streptozotocin and with other normal rats maintained on a restricted diet to obtain a daily body weight change similar to that of the diabetics. In diabetic rats plasma levels of Val, Ile and Leu rose and those of Asp, Thr, Ser, Gln, Gly, Tyr, Orn, Lys, His and Arg decreased, whereas brain concentrations of Leu, Arg and Orn were augmented and those of Thr and Ser reduced. Insulin treatment diminished these differences in comparison with controls values. In food-restricted normal rats plasma and brain amino acid concentrations also differed from values in normal controls but they were of different magnitude and/or direction than those of diabetics. In vitro 3H-Leucine incorporation into proteins by brain postmitochondrial dialyzed supernatants was unaffected in both diabetic and food-restricted rats, whereas in liver preparations the same parameter was significantly reduced in both groups and insulin treatment of the diabetics decreased this difference. Results indicate that brain amino acid concentrations in diabetic animals are a secondary consequence of their circulating levels and of potential modifications of brain amino acid metabolism other than protein synthesis, which is unaffected.

Amino Acids↗

PubMed1981

Tryptophan overload in the pregnant rat: effect on brain amino acid levels in in vitro protein synthesis.

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of [3H]leucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.

Amino Acids↗

PubMed1980

Age-dependent changes in brain protein synthesis in the rat.

Brain protein synthesis was studied in vivo, in brain slices, and in cell-free systems in rats aged 1, 16, and 24 months. We observed a highly significant reduction in amino acid incorporation with advancing age. This reduction was observed in vivo, in slices, in postmitochondrial supernatant, microsomes, and membrane-bound polysomes. Free heavy polysomes showed no age-dependent decline but formed a smaller proportion of total ribosomes in older animals. These studies suggest that in the rat brain protein synthesis declines before senescence, possibly due to an impairment in the initiation process.

Age Factors↗

PubMed1980

A simple reproducible cell-free system for measuring brain protein synthesis.

A simple, rapid, sensitive, and reproducible cell-free assay system for studying brain protein synthesis is described. This system uses small amounts of brain postmitochondrial supernatant, making it a convenient screening test when only small amounts of tissue are available. It showed over 95% dependence on Mg2+ and on an energy source. Optimal incorporation occurred under the following conditions: Mg2+ 3 mM; ATP, 0.6 mM; GTP, 0.6 mM; high K+, greater than or equal to 25 mM; Low Na+, less than or equal to 15 mM; pH 7.1-7.5. The rate of amino acid incorporation did not vary with leucine concentrations in vitro up to 1 mM, which obviated the need to measure endogenous leucine concentrations.

Adenosine Triphosphate↗

PubMed1980

Rat brain protein synthesis declines during postdevelopmental aging.

Using improved methods to measure brain protein synthesis in vivo (Dunlop et al., 1975) we have established that brain protein synthesis significantly declines in forebrain, cerebellum and brain stem when mature rats (3 months old) are compared to old rats (22.5 months old). The incorporation of (3H) L-lysine into forebrain protein is reduced 11% in 10.5 month old rats compared to 3 month old rats. A further reduction of 9% occurred between 16.5 months and 22.5 months. Our data suggest that reduced levels of protein synthesis initiation may be responsible, at least in part, for this age-related decline.

Aging↗

PubMed1979