Inhibited Leucine Incorporation Research Articles

Action of ethionine, methionine, ATP, adenine, albumin and Ca 2+ on protein synthesis of normal and tumor cells

In the experiments here reported it has been found that ethionine as well as methionine affect leucine incorporation into protein of hepatoma cells to about the same extent. In vitro the effects of methionine on leucine incorporation into liver protein are less than those of ethionine. The effects of ethionine are the same in liver of male or female rats. The incorporation of amino acids other than leucine into protein of liver slices is also inhibited either by ethionine or by methionine. In vivo ethionine but not methionine inhibit leucine incorporation into total liver protein. ATP strongly affects leucine incorporation into protein. Also in the absence of Ca 2+ leucine incorporation into normal tissues and hepatoma cells is greatly reduced. A different response among the tissues to Ca 2+ variations has been found. The addition of Ca 2+ slightly counteracts the effects of ATP. Adenine stimulates leucine incorporation in hepatoma cells but not in liver slices. Adenine slightly counteracts the effects of ethionine. At physiological concentrations albumin inhibits leucine incorporation in normal tissues and hepatoma cells, but at lower concentrations the response can differ. In the absence of Ca 2+ and Mg 2+ albumin depresses leucine incorporation in liver but stimulates it in hepatoma cells.

Effect of inhibition of mitochondrial protein synthesis in vitro upon thyroxine stimulation of oxygen consumption.

It has been suggested that the calorigenic activity of thyroid hormones is secondary to their ability to stimulate mitochondrial or ribosomal protein synthesis. Since thyroid hormones stimulate both protein synthesis and oxgyen consumption in mitochondiia from normal rats, the effect of inhibition of protein synthesis upon thyroxine (T4) stimulation of mitochondrial oxygen consumption in vitro was examined. T4 (50 JUM) stimulated leucine incorporation 125% and oxygen consumption 100% (without ADP) in mitochondria from normal rats in vitro. The effects were linear over 8 min. Chloramphenicol (250 /xM) or puromycin (100 fjM) inhibited leucine incorporation 85–91% in control and T4-containing reactions without affecting oxygen consumption in either. ADP/O and respiratory control ratios were significantly depressed by T4; however, the antibiotics had no effects on these values, with or without T4. In addition, T4 was found not to alter the rate of decay of mitochondrial protein, newly formed in vitro. (Endocr...

Control of Protein and RNA Synthesis by AMO-1618 and other Growth Retardants in Cucumber Cotyledons

Cotyledons dissected from 5 day old etiolated seedlings of cucumber were incubated with growth regulators for 21 or 45 hours in light and then fed with leucine- 14 C or uracil- 14 C; chlorophyll, protein and RNA were estimated. 1. AMO-1618 (0.001 M) inhibited growth and chlorophyll synthesis by about 50 %; the effects were completely reversed by KCl (0.025 M) and reduced by benzylaminopurine (BAP, 10 −5 M) or gibberellic acid (GA 3 , 10 −4 M). Other growth retardants, CCC (0.005 M), Phosfon D (10 −4 M) vnd B-Nine (0.01 M) decreased chlorophyll synthesis by about 50% with a little effect on growth 2. In the control cotyledons, incubated in light for 1 day, the level of protein was reduced and that of RNA increased from original 1.12 mg and 93 μg to 1.08 mg and 138 μg per cotyledon basis, respectively; incorporation of leucine- 14 C into protein and uracil- 14 C into RNA was increased, in comparison with the original cotyledons, by about 6 times. 3. KCl stimulated protein synthesis by about 250 per cent in comparison with the water treated control, and slightly reduced RNA sythesis. GA 3 and BAP transiently increased protein and RNA synthesis, respectively. Under the influence of BAP the content of RNA was increased to 206 μg/cotyledon. 4. AMO-1618 (0.001 M), B-Nine (0.01 M) and Phosfon D (10 −4 M) in 1 day tests inhibited leucine incorporation into protein by about 50 %. The retardants reduced a rise in RNA content. AMO-1618 had little effect on uraci- 14 C incorporation into RNA, the other 2 growth retardants inhibited this process. CCC at 0.005 M concentration inhibited RNA synthesis being without effect on protein synthesis; CCC at a higher concentration of 0.01 M or in 2 day lasting experiments also reduced protein synthesis. The effects of growth retardants on RNA synthesis were reduced by BAP and those on protein synthesis by KCl. CCC completely erased the stimulatory effect of GA 3 on protein synthesis. 5. B-Nine intensified leucine and uracil uptake from the incuhational media; the same was true for KCl. It is concluded that plant growth retarding chemicals inhibit chlorophyll synthesis in consequence of inhibition of protein (AMO-1618, B-Nine, Phosfon D) or RNA (CCC, Phosfon D) metabolism in detached cucumber cotyledons. K + -induced growth and chlorophyll synthesis follows the selective stimulation of protein synthesis.

Reactivation of ribonucleic acid and protein synthesis during germination of Blastocladiella zoospores and the role of the ribosomal nuclear cap.

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.

Inhibition of protein synthesis in ehrlich ascites-tumour cells by the phenanthrene alkaloids tylophorine, tylocrebrine and cryptopleurine

The phenanthroindolizidine alkaloids tylophorine and tylocrebrine and the phenanthroquinolizidine alkaloid cryptopleurine inhibit incorporation of leucine into protein in Ehrlich ascites-tumour cells by 50% at 1 × 10 −6 M, 2 × 10 −7 M, and 2 × 10 −8 M respectively, but do not inhibit incorporation of uracil into nucleic acids at 10 −5 M. Cryptopleurine inhibits leucine incorporation into protein in a cell-free system from Ehrlich cells, but at 10 −5 M does not inhibit protein synthesis in Escherichia coli.

Metabolic effects of hypoglycemic sulfonylureas—I: In vitro effect of sulfonylureas on leucine incorporation and metabolism and on respiration of rat tissues

The hypoglycemic sulfonylureas: chlorpropamide, tolbutamide, carbutamide and glycodiazine were found to inhibit leucine incorporation into the protein of rat diaphragm, liver, kidney and adipose tissue. This inhibition was not the result of a reduced amino acid transport, an alteration in the metabolism of leucine or an impaired endogenous respiration of these tissues. A stimulation of oxygen uptake was observed in muscle and liver early during the incubation. Glucose and insulin relieve to some extent the inhibition of leucine incorporation by sulfonylureas and this could indicate that the inhibition of protein synthesis is related to an impaired energy metabolism. The oxidative decarboxylation of leucine, in the four tissues studied, is strongly inhibited by chlorpropamide and is mainly responsible for the increased cellular levels of leucine found in diaphragm after incubation with chlorpropamide and tolbutamide. The possibility of a link between sulfonylurea- and leucine hypoglycemia is discussed.

Similarities between the biochemical actions of cycasin and dimethylnitrosamine.

1. Rats were given the hepatotoxin and carcinogen cycasin by stomach tube. In one experiment, rats whose RNA had previously been labelled with [(14)C]-formate were given the acetate ester of the aglycone form of cycasin, methylazoxymethanol, by intraperitoneal injection. 2. Incorporation of (14)C from l-[U-(14)C]leucine into the proteins of some organs was measured in cycasin-treated rats. Cycasin inhibited leucine incorporation into liver proteins but not into kidney, spleen or ileum proteins. This inhibition was not evident until about 5hr. after cycasin administration, but once established it persisted for the next 20hr. 3. Methylation of nucleic acids was detected in some organs of rats treated with cycasin or methylazoxymethanol. The purine bases of RNA and DNA were isolated by acid hydrolysis followed by ion-exchange column chromatography. The resulting chromatograms showed an additional purine base that was identified as 7-methylguanine. It was shown that, in animals treated with the toxin, liver RNA was methylated to a greater extent than was either kidney or small-intestine RNA. Also, as a result of cycasin administration, liver DNA guanine was methylated to a greater extent than was RNA guanine. 4. These results are discussed in relation to comparable experiments with dimethylnitrosamine. It is suggested that cycasin and dimethylnitrosamine are metabolized to the same biochemically active compound, perhaps diazomethane, but that various tissues differ in their capacity to metabolize the two carcinogens.