Evidence for cycloheximide acting as a glutamine analogue in plant tissue.

In growing maize root tissue [14C]asparagine formation in inhibited and [14C]glutamine accumulation stimulated by treatment with cycloheximide or glutamine analogs such as azaserine. In contrast, puromycin enhances the accumulation of [14C]asparagine but not [14C]glutamine. Cycloheximide and puromycin alone inhibit protein synthesis. This is interpreted to mean that the alteration in amide metabolism following cycloheximide treatment is a direct result of the antibiotic acting as a glutamine analog. While cycloheximide is often the cytoplasmic protein synthesis inhibitor of choice due to its potency and rapid action, its assumed specificity of action of eukaryotes is doubtful.     

Effects of adrenocorticotropin and cycloheximide on adrenal diglyceride kinase

We studied the effects of adrenocorticotropin (ACTH) and cycloheximide on adrenal enzymes involved in phosphatidate synthesis. Treatment of rats in vivo with ACTH induced a rapid increase in phosphatide synthesis from diglyceride and ATP in adrenal homogenates, and cycloheximide treatment prevented this increase if given before ACTH and rapidly reversed the increase if given after ACTH. The stimulatory effect of ACTH appeared to be largely due to an increase in diglyceride substrate, as kinase activity was not altered. The inhibitory effect of cycloheximide, on the other hand, appeared to be due to a decrease in diglyceride kinase activity. Neither ACTH nor cycloheximide treatment had any effect on the activity of glycerol-3'-phosphate acyltransferase or phosphatidate phosphatase. Our findings suggest that (a) ACTH increases the flow of phospholipid (and their levels) throughout the entire circular pathway, i.e., phosphatidate leads to CDP-diacylglycerol leads to inositides leads to diglycerides leads to phosphatidate, and (b) a labile protein may serve to allow entry into a recycling of diglyceride in this pathway. In addition, since cycloheximide blocked carbachol-induced increases in pancreatic and salivary glandular phosphatidate synthesis resulting from phosphatidylinositol hydrolysis and consequent diglyceride generation, the putative labile protein may have widespread importance.     

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina.

1. The rates of detoxification of cycloheximide (33 mug/g fresh wt.), puromycin (167 mug/g fresh wt.) and actinomycin D (1 mug/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

Stimulation by glucagon and adenosine-3',5'-cyclic monophosphate of protein degradation in Reuber H35 hepatoma monolayers

Protein degradation in Reuber H35 hepatoma monolayers was measured as release of radioactive trichloroacetic acid-soluble material from intracellular protein labelled with [3H]leucine for 16 hr followed by 3-hr chase period. Proteolysis in this system was stimulated by physiological concentration of glucagon reaching a maximum at 10(-7) M with an increase of 30%. Dibutyryl cyclic AMP also had a stimulatory effect. When both glucagon and dibutyryl cyclic AMP were present at optimal concentrations, their effects were not additive suggesting that glucagon may act via the formation of cyclic AMP. In the presence of protein synthesis inhibitor, cycloheximide or puromycin, proteolysis remained responsive to glucagon. Glucagon counteracted the inhibitory effect of insulin on proteolysis.     

Corticotropin-induced changes in a soluble desmolase preparation

Following simple homogenization, substantial desmolase activity is recovered in rat adrenal 105 000 × g supernatant. The desmolase complex sediments at 3–4 S on sucrose gradients, is found in the clear cytosol, requires NADPH, is derived from mitochondria and is inhibited by aminoglutethimide and pregnenolone. The lipid fraction contains little or no desmolase activity but greatly enhances pregnenolone synthesis in soluble desmolase preparations, presumably by supplying free cholesterol substrate. Prior adrenocorticotropin (ACTH) administration enhances pregnenolone synthesis in the 105 000 × g supernatant, and cycloheximide, an inhibitor of adrenal protein synthesis, does not block this effect of ACTH (but rather potentiates it). The ACTH effect may be largely explained by an increase in free cholesterol, which enhances the activity of both the lipid fraction and clear cytosol, since: free cholesterol levels are increased by ACTH, particularly with cycloheximide pretreatment; type I and inverted type I difference spectrum changes, indicating greater cholesterol availability for binding to cytochrome P-450, are enhanced by ACTH with or without cycloheximide treatment; cholesterol-rich lipid fraction enhances such spectral changes and obliterates the differences in spectral and pregnenolone-synthesizing activities betwen control and ACTH-stimulated soluble desmolase preparations; and desmolase stimulatory properties of clear cytosol co-chromatographs with [¹⁴C]cholesterol. Since cycloheximide blocks ACTH-induced effects in intact mitochondria but not in the soluble desmolase preparation, it is postulated that the labile protein required during ACTH action functions to overcome a ?restraining influence’ which is present in intact mitochondria but not in the soluble desmolase system. The ‘restraining influence’ may be due to limited cholesterol-desmolase interaction.     

Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway.

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2--3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.     

Further observations on the increases in inositide phospholipids after stimulation by ACTH, cAMP and insulin, and on discrepancies in phosphatidylinositol mass and 32PO4-labeling during inhibition of hormonal effects by cycloheximide

ACTH-induced increases in adrenal polyphosphoinositides were demonstrable after extraction by acid or non-acid methods, and after purification by unidimensional or two-dimensional thin layer chromatography. ACTH-induced increases in phosphatidylinositol mass were apparent, both as increases in measurable phosphorus and inositol. ACTH and insulin also increased 32PO4 incorporation into adrenal and adipose tissue polyphosphoinositides which were acid-extracted and purified by two-dimensional chromatography. Cyclic-AMP increased the mass of phosphatidylinositol, but decreased 32PO4 incorporation into this and other phospholipids; the cause for this decrease in specific activity was not evident. Increasing doses of cycloheximide progressively inhibited ACTH- or insulin-induced increases in the mass of phosphatidylinositol, but 32PO4 incorporation therein followed a bimodal curve, with inhibition at lower doses and stimulation at higher doses; these divergent changes in phosphatidylinositol mass and 32P-labeling at higher concentrations raise the possibility that cycloheximide may activate phospholipases in hormone-stimulated tissues. These results offer further support for our contention that ACTH and insulin increase inositide phospholipid concentrations in their target tissues by a cycloheximide-sensitive mechanism.     

The relation of protein synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

The effect of cycloheximide on chondroitin sulphate biosynthesis was studied in bovine articular cartilage maintained in culture. Addition of 0.4 mM-cycloheximide to the culture medium was followed, over the next 4h, by a first-order decrease in the rate of incorporation of [35S]sulphate into glycosaminoglycan (half-life, t 1/2 = 32 min), which is consistent with the depletion of a pool of proteoglycan core protein. Addition of 1.0 mM-benzyl beta-D-xyloside increased the rate of incorporation of [35S]sulphate and [3H]acetate into glycosaminoglycan, but this elevated rate was also diminished by cycloheximide. It was concluded that cycloheximide exerted two effects on the tissue; not only did it inhibit the synthesis of the core protein, but it also lowered the tissue's capacity for chondroitin sulphate chain synthesis. Similar results were obtained with chick chondrocytes grown in high-density cultures. Although the exact mechanism of this secondary effect of cycloheximide is not known, it was shown that there was no detectable change in cellular ATP concentration or in the amount of three glycosyltransferases (galactosyltransferase-I, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II) involved in chondroitin sulphate chain synthesis. The sizes of the glycosaminoglycan chains formed in the presence of cycloheximide were larger than those formed in control cultures, whereas those synthesized in the presence of benzyl beta-D-xyloside were consistently smaller, irrespective of the presence of cycloheximide. These results suggest that beta-D-xylosides must be used with caution to study chondroitin sulphate biosynthesis as an event entirely independent of proteoglycan core-protein synthesis, and they also indicate a possible involvement of the core protein in the activation of the enzymes of chondroitin sulphate synthesis.     

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.     

The effect of D-xylose, beta-D-xylosides and beta-D-galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage.

The incorporation of [3H]acetate into chondroitin sulphate was used as a measure of the rate of synthesis of this polysaccharide in whole tibias and femurs of embryonic chicken cartilage in vitro. The incorporation is inhibited by puromycin and by cycloheximide, but the inhibition is relieved by the addition of D-xylose, beta-D-xylosides and beta-D-galactosides to the incubation medium. Beta-D-Xylosides can stimulate the incorporation to 300% of that of controls incubated in the absence of cycloheximide or puromycin, D-Xylose, beta-D-xylosides and beta-D-galactosides appear to act as artificial initiators of chondroitin sulphate synthesis and enable polysaccharide-chain synthesis to be studied as an event separate from the synthesis of intact proteoglycan.     

Melanoprotein: absence of a direct melanin-protein relationship in chick embryo melanocytes.

Short-term synthesis of radioactivity labeled melanin (using DL-[2-14C]tyrosine or 2-[2-14C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cycloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in L-[U-14C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in DL-[2-14C]tyrosine or 2-[2-14C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.     

Aminoacyl transfer from phenylalanyl-tRNA microinjected into Xenopus laevis oocytes.

$\text{Xenopus laevis}$ oocytes were injected with [¹⁴C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [¹²C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.     

THE EFFECT OF PUROMYCIN ON INTRANUCLEAR STEPS IN RIBOSOME BIOSYNTHESIS

Inhibition of protein synthesis by puromycin (100 γ/ml) is known to inhibit the synthesis of ribosomes. However, ribosomal precursor RNA (45S) continues to be synthesized, methylated, and processed. Cell fractionation studies revealed that, although the initial processing (45S → 32S + 16S) occurs in the presence of puromycin, the 16S moiety is immediately degraded. No species of ribosomal RNA can be found to have emerged from the nucleolus. The RNA formed in the presence of puromycin is normal as judged by its ability to enter new ribosomal particles after puromycin is removed. This sequence of events is not a result of inhibition of protein synthesis, for cycloheximide, another inhibitor of protein synthesis, either alone or in combination with puromycin allows the completion of new ribosomes.     

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light ( Hartwell et al. 1996 ; Plant Journal 10 , 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μ M ) strongly inhibited the light-induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L -malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light-induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 m M puromycin or 100 μ M cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light-induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.