Short term labeling of proteins,gangliosides and glycoproteins of the optic tract of chickens exposed to light or darkness

In contrast with previous findings of the labeling of the glycosidic moieties of the gangliosides and glycoproteins in chickens injected with $\text{N-[}{}^3\text{H}\text{]}\text{acetylmannosamine}$, the labeling of the ganglion cell layer and optic tectum proteins of chicks exposed to light after an intraocular injection of [³H]proline showed no differences with those of their counterpart chickens that remained in darkness. The same failure in finding a difference was met when the cytosolic or the particulate proteins or the acid soluble fraction in the retina were compared.Cycloheximide and puromycin inhibited the labeling of retina and optic tectum proteins, gangliosides and glycoproteins in both illumination conditions. Since the labelings in the optic tectum appeared more inhibited than those in the retina ganglion cell layer it was concluded that cycloheximide and puromycin, besides the synthesis of those compounds, also inhibit their axonal transport.On the basis of these contrasting results the working hypothesis is advanced that light stimulation enhances the activity of the Golgi apparatus but not (or less) that of the polyribosomes.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

Incorporation of radioactive shikimic acid into N-(γ-Lglutamyl)-4-hydroxyaniline in Agaricus bisporus

Agaricus bisporus contains novel aromatic compounds. By incubation of the mushroom with [G-¹⁴ C] shikimic acid, the radioactivity was incorporated into tyrosine, phenylalanine and several unidentified metabolites. The most radioactive metabolite in the stipe and the cap was identified as $\text{N-(γ-}\text{L}\text{-glutamyl}\text{)-4-}\text{hyrroxyaniline}$. The radioactivity was proved to be localized in the 4-hydroxyaniline moiety of this compound.     

The secretion of parathormone and glycosylated proteins by parathyroid cells in culture

The secretion of radioactive peptides by dispersed porcine parathyroid cells incubated with [³H]- or [¹⁴C]amino acids, [³H]glucosamine and [³H]mannose was analyzed. After incubation, the culture medium contained radioactive parathormone, as expected, and two radioactive glycopeptides: SP I and SP II. SP I appears to be identical with $\text{parathyroid}\text{secretory}\text{protein}$, heretofore not recognized as a glycoprotein. SP II has not been previously identified. SP I, but not SP II or parathormone, was adsorbed by Concanavalin A possibly reflecting a high mannose content of this molecule. Raising the concentration of calcium in the medium suppressed the secretion of radioactive parathormone and SP I in a similar fashion but did not affect the secretion of SP II. Our results suggest that SP I may play a fundamental role in parathyroid synthetic or secretory processes.     

Ozonolytic cleavage of authentic and pancreatic dolichyl mannopyranosyl phosphates: determination of sugar configuration in the fragments with alpha- and beta-mannosidases.

Exposure of authentic dolichyl α-D-[¹⁴C]mannopyranosyl phosphate ($\text{I}$) or calf pancreas dolichyl [¹⁴C]mannopyranosyl phosphate ($\text{II}$) to ozone at ?70° in pentane followed by treatment with triphenylphosphine gave water-soluble fragments in 65–95% yield. The radioactive products obtained were similar; the major fragment had a mobility on tlc greater than that of mannose but lower than that of citronellyl β-D-mannopyranosyl phosphate. The electrophoretic behavior of the fragments indicated that they possessed intact phosphodiester linkages. α-Mannosidase released [¹⁴C]mannose from the fragments of $\text{I}$ but not from the fragments of $\text{II}$; however, the latter were susceptible to β-mannosidase indicating that the pancreatic mannolipid contains a β-linked mannosyl residue.     

Energization of phenylalanine transport and energy-dependent transhydrogenase by ATP in cytochrome-deficient Escherichia coli K12

$\text{E. coli}$ SASX76 does not form cytochromes unless supplemented with 5-aminolevulinic acid. Uptake of [¹⁴C]phenylalanine into cytochrome-deficient cells of this mutant was energized by glucose but not by endogenous substrates or D-lactate with or with or without fumarate. In contrast, uptake of this amino acid was supported in cytochrome-containing cells of this strain by oxidation of D-lactate or endogenous substrates. It is concluded that ATP can energize phenylalanine transport in cytochrome-deficient cells. Cytochrome-deficient cells lacked eńergy-dependent transhydrogenase activity driven by oxidation of NADH but ATP-driven transhydrogenation was unimpaired. Both transhydrogenase activities were present in cytochrome-containing cells.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

Incorporation of cinnamic acid-2-[14C] into tylophorine

Tylophora indica plants have been shown to contain phenanthroindolizidine alkaloids of the tylophorine type. Cinnamic acid-[2-¹⁴C]was incorporated efficiently into these alkaloids supporting the hypothesis that ring A and C-10 and C-6$\text{\$}\text{?}$of tylophorine are derived from phenylalanine.     

Modification of transfer ribonucleic acid by an alkylating fragment from S-(1,2-dichlorovinyl)-L-cysteine

An alkylating fragment derived by enzymatic cleavage of [³⁵S]-(1,2-dichlorovinyl)-L-cysteine reacted, apparently covalently, with RNA isolated from $\text{E. coli}$, and from livers of the bovine calf, rat and rabbit. Transfer RNA was much more susceptible to alkylation than ribosomal RNA as revealed by gel filtration technique, and measurement of [³⁵S] substitution into nucleotides. Unfractionated $\text{E. coli}$ tRNA modified by such reaction accepted most amino acids to the same extent as control tRNA, although about 40% less acceptance was observed for L-histidine, L-serine and L-tyrosine. Study of ribosomal binding, however, indicated an impairment of codonanticodon interaction between synthetic polynucleotide messengers and amino acyl substituted, alkylated tRNA.     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Anomalies in 5'-end [32P] labelling of transfer RNA and partial sequence of a tRNAGlu from Scenedesmus obliquus

A chloroplast tRNA_m^Met species from $\text{Scenedesmus}\text{obliquus}$ is very poorly 5′-end [${}^{32}\text{P}$] labelled using [γ-³²P]ATP and T4 polynucleotide kinase. In sequencing the tRNA using standard 5′-labelled methods a very minor contaminating tRNA is preferentially labelled. The partial tRNA sequence determined by this method has an anticodon (CUC) for tRNA^Glu.     

Synthesis and glycosylation of the MOPC-46B immunoglobulin in kappa chain in Xenopus laevis oocytes.

Polyadenylated mRNA isolated from MOPC-46B plasmacytoma, which secretes a glycosylated kappa chain, was injected into $\text{Xenopus laevis}$ oocytes. Analysis of the resulting product showed that [1-¹⁴C]mannose was incorporated into the MOPC-46B kappa chain. Light chains synthesized in oocytes injected with mRNA from MOPC-321 plasmacytoma, which secretes a nonglycosylated kappa chain, failed to incorporate label from [1-¹⁴C]mannose. Thus, protein glycosylation in the oocyte is apparently specific in that carbohydrate is incorporated only into the kappa chain synthesized as a glycoprotein by myeloma cells. It is thus evident that the general signals for glycosylation have remained stable during independent evolution of the amphibia and mammalia.     

Biosynthesis of -linolenic acid by disrupted spinach chloroplasts

A disrupted spinach chloroplast preparation readily synthesized $\text{[}{}^{14}\text{C]α-linolenate}$ from [2-¹⁴C]acetate under anaerobic conditions. It can be shown by degradation data that [¹⁴C]oleate is not a precursor of [¹⁴C]linolenate and that $\text{cis}$ 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [¹⁴C]linolenate.     

The importance of transamination and decarboxylation in phenylalanine metabolism in vivo in the rat

The extent of hydroxylation, transamination, and decarboxylation in the metabolism of excess phenylalanine in vivo has been examined by measuring the amount of radioactivity from [¹⁴C]phenylalanine that is converted to ¹⁴CO₂ and urinary metabolites. Transamination and direct decarboxylation represent only 6% of total phenylalanine metabolism. The major aromatic metabolites in the urine after phenylalanine loading are phenylacetylglycine, phenylacetic acid, phenylpyruvate, and phenylalanine. A small but significant portion (1.5%) of phenylalanine is degraded to nonaromatic compounds. The maximum phenylalanine oxidation in vivo is approximately $\text{75\%}\text{24}$ h at saturating concentrations of phenylalanine; thus, the major route of degradation of phenylalanine in the rat, even when intake is high, is via formation of acetoacetic acid and fumaric acid.     

Methylation of transfer RNA during transformation of human lymphocytes by phytohemagglutinin

Methylation of transfer RNA during phytohemagglutinin induced transformation of human lymphocytes was studied by labeling the tRNA $\text{in}\text{vivo}$ with (methyl-H³)-methionine and measuring the distribution of tritium in the methylated nucleotides. An alteration in the pattern of methylation occurred within hours after PHA-stimulation and this change was maintained through several cell generations. There was a 50 to 94% increase in the relative amount of methylated N²-methyl-guanine (1 to 3 hr) and a 40 to 59% decrease in the relative amount of 1-methyladenine (1 to 12 hr). Treatment of the stimulated cells with Actinomycin D prevented the subsequent methylation not tRNA. However, inhibition of protein synthesis by puromycin did not effect the early changes observed in the methylation of tRNA.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

Protein synthesis in cultured muscle cells: methylation of nascent proteins

Protein methylation was examined in primary cultures of rat leg muscle cells between 7 and 9 days of culture. Methyl[¹⁴C]- or [³H]-methionine was introduced into the culture medium and the cells were sampled for radioactive methylated protein residues. Incorporation of the total radioactivity was linear for at least 4 hr after introduction of the methionine label. When labeling was studied for periods between 10–30 min, the methylation of polyribosome-bound, presumably nascent, proteins was unaffected by addition of cycloheximide to the culture medium. The antibiotic, however, inhibited incorporation of methionine, and consequently increased the ratios of the incorporated methylated, to methionine residues and the ratio of ribosome-bound to free radioactivity. The methylated, polyribosome-bound proteins were decreased when puromycin was added to the culture medium. It is proposed that selective methylation of nascent proteins, such as myosin, can begin at the level of polyribosomes and be completed in the cytosol of muscle cells cultured in vitro.     

Biosynthesis of proteins, nucleic acids and glycosphingolipids by synchronized KB cells

The biosynthesis of glycosphingolipids and various types of proteins and nucleic acids at specific periods of the cell cycle was studied by using synchronized KB cells. Maximum incorporation of radioactive galactose, leucine and thymidine into several proteins and nucleic acids occurred as has been reported previously (6,11). Maximum incorporation of $\text{D}$-1[¹⁴C] galactose into glycosphingolipids was observed during the M and G-1 phases. There was a 5 fold increase in the levels of gangliosides and combined neutral glycosphingolipids during the M and G-1 phases. Thus, regulated biosynthesis of glycosphingolipids and macromolecules might be important in the cyclic expression of some of the functional properties which are characteristic of these compounds.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.