Low Concentrations Of Cycloheximide Research Articles

Indomethacin inhibits viral RNA and protein synthesis in cells infected with vesicular stomatitis virus

Treatment of HEp-2 cells with 10 −3 M indomethacin prior to infection with vesicular stomatitis virus (VSV) blocked synthesis of viral RNA species and proteins. Host cell macromolecular synthesis was also inhibited by this antagonist of prostaglandin metabolism. The reversibility of this drug-mediated inhibition was a time-dependent process that could be prevented after removal of indomethacin if cells were exposed to low concentrations of cycloheximide which do not normally interfere with VSV replication. That indomethacin exerts its inhibitory effect at an early stage of infection is suggested by the finding that this drug can compromise viral transcription either in vitro or in vivo.

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Synrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G 2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G 2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.

Differential kinetics of changes in the state of phosphorylation of ribosomal protein S6 and in the rate of protein synthesis in MPC 11 cells during tonicity shifts.

Mouse myeloma (MPC 11) cells respond rapidly to hypertonic conditions by shutting down protein synthesis at the level of polypeptide chain initiation. Translational activity recovers equally quickly upon a return to isotonicity. Disaggregation and reformation of polysomes occur in parallel to the changes in protein synthesis. Ribosomal protein S6 becomes dephosphorylated under hypertonic conditions and rephosphorylated when isotonic conditions are restored. The kinetics with which these changes occur are, however, too slow to account for the changes in protein synthesis. Treatment of the cells with a low concentration of cycloheximide allows reformation of polysomes under hypertonic conditions; conversely, puromycin prevents the restoration of polysomes which otherwise occurs on return to isotonicity. Neither inhibitor prevents the changes in S6 phosphorylation resulting from the tonicity shifts. We conclude that the overall extent of phosphorylation of S6 neither regulates nor is determined by the rate of protein synthesis and is not obligatorily related to the proportion of ribosomes in polysomes.

Regulation of hexose carriers in chicken embryo fibroblasts. Effect of glucose starvation and role of protein synthesis.

Regulation of hexose transport was investigated in chicken embryo fibroblasts (CEF) which develop 4- to 8-fold enhanced hexose transport activity during glucose starvation. The presence of cycloheximide in low (0.5 micrograms/ml) concentrations during starvation largely blocked the enhancement of transport activity. Glucose refeeding of CEF in the starvation state led to a decline in transport to the basal level. This decline was either potentiated or blocked by the presence of cycloheximide in low or high (50 micrograms/ml) concentrations, respectively. Exposure of CEF in the fed state to low concentrations of cycloheximide resulted in a 70% decrease of transport within 6 h, whereas exposure to high concentrations of cycloheximide led to only a modest loss (35% decrease). In the glucose-starved state, CEF had no significant decline of transport when exposed to cycloheximide at either high or low concentrations. The uptake of 3-O-methylglucose by fed, starved, or cycloheximide-treated CEF correlated closely with D-glucose transport activity and [3H]cytochalasin B binding by plasma membranes prepared from CEF exposed to the same conditions. Hexose transport activity of CEF seems to largely depend on the number of functioning carriers in the plasma membrane, which apparently reflect the balance between carrier synthesis and inactivation. These two processes require protein synthesis, but are differentially sensitive to the effects of cycloheximide, such that low concentrations of cycloheximide appear to block primarily synthesis while high concentrations block both processes. Furthermore, during starvation the enhancement of transport appears largely due to decreased carrier inactivation in the face of continued carrier synthesis.

Butyrate inhibits mouse fibroblasts at a control point in the G1 phase.

Butyrate block 3T6 cells in the G1 phase of the cell cycle approximately 5--6 h prior to the start of the S phase. Serum factors are required before as well as after the butyrate-sensitive steps in G1 in order to allow cells to start DNA synthesis. 3T6 cells infected with SV40 or with polyoma virus are also blocked at the same stage in G1 in the presence of the fatty acid. However, events before as well as after the butyrate-sensitive step do not require serum in virus-infected cells. The sensitivity of the initiation of cellular DNA synthesis to increasing concentrations of butyrate is the same for serum-stimulated or for virus-infected cells. A similar and parallel effect on DNA synthesis is observed if cells are incubated in the presence of very small amounts of cycloheximide. After release of the cycloheximide-induced G1 arrest about 4--6 h have to pass before cells enter the S phase. Cells stably transformed by SV40 are considerably more resistant to low cycloheximide concentrations and to butyrate. These data are discussed in the light of the hypothesis that both low concentrations of cycloheximide and sodium butyrate block cells at a control point in G1 by interference with the synthesis of one or more rapidly turning over, cell cycle-specific proteins.

Messenger RNA species partially in a repressed state in mouse sarcoma ascites cells.

Four major mRNA species of mouse sarcoma ascites cells, coding for polypeptides designated P65, P40, P36, and P21, occur predominantly as untranslated messenger ribonucleoprotein particles. Cloned cDNA probes were used to study their distribution in cytoplasmic extracts of these cells. A considerable portion of the mRNA molecules sedimented as small particles, whereas the rest was present in polyribosomes. In contrast, the actin mRNA was present almost exclusively in polyribosomes. Incubation of the ascites cells in culture medium, particularly after a starvation treatment, caused an enhancement in polypeptide chain initiation relative to elongation in these cells, as evidenced by a shift of ribosomes into the polyribosome fraction and by an increase in polyribosome size. Exposure of the cells to a low concentration of cycloheximide, an inhibitor of the elongation step, had a similar effect. The actin mRNA and the active P65, P40, P36, and P21 mRNA molecules were shifted to larger polyribosomes in the treated cells, but no shift of molecules from small particles to polyribosomes was observed. The incubation in culture also led to considerable increases in the proportion of P65 and P40 mRNA molecules in the untranslated state. The results indicate that the untranslated state cannot be attributed to poor initiation efficiency. It is suggested that a portion of the mRNA molecules is maintained in a repressed state and that mRNA repression may represent an important translation control process.

Sexual development in a homothallic fission yeast: synthesis of readiness proteins resolved by gel electrophoresis.

Sexual development of a homothallic strain of Schizosaccharomyces pombe was monitored by radiolabelling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Of more than 60 bands detected by Coomassie brilliant blue and by autoradiography, about 30 bands synthesized during development were discrete enough for experimental analysis. About a dozen bands are preferentially vegetative, another dozen preferentially developmental. However, vegetative bands as a group are also synthesized during development. Their synthesis is relatively unaffected by low concentrations of cycloheximide or by chloramphenicol and is not temperature sensitive at 37 degrees C nor catabolite repressible. Only band 40 (ca. 40 000 daltons) seems to be exclusively vegetative. The synthesis of developmental bands 13, 18, 24, 30, and alpha, all of which first appear during late-log phase, is catabolite repressible. Developmental band 51 is also synthesized throughout the vegetative phase. The synthesis of bands 24, 30, 51, and alpha is temperature sensitive at 37 degrees C during the development, but that of band 18 is not. The synthesis of band 13 during development is not temperature sensitive, but its earlier synthesis during late-log phase is. The synthesis of all these six developmental bands is immediately inhibited by cycloheximide, but not by chloramphenicol. Their appearance as a group of radioactive bands is greatly diminished in cultures grown in cycloheximide, in chloramphenicol, or in ethidium bromide. Developmental bands 13, 18, 24, and 30 may be called readiness proteins. They first appear prior to the earliest morphological signs of sexual activity. Their developmental synthesis is inhibited by conditions that inhibit sexual development. Such inhibitory conditions include anaerobiosis, restrictive temperature, aging in stationary phase, the presence of inhibitors of cytoplasmic protein synthesis and of mitochondrial function, and catabolite repression. Readiness proteins may be regulating the switch from vegetative metabolism.

Investigation of the role of uncharged tRNA in the regulation of polypeptide chain initiation by amino acid starvation in cultured mammalian cells; a reappraisal.

The rate of initiation of protein synthesis in mammalian cells in culture is regulated by the availability of essential amino acids. Previous reports have suggested that this effect is mediated by changes in the ratio of uncharged to charged tRNA species in these cells. We have investigated this possibility by examining the formation of 40‐S subunit. Met‐tRNAf ribosomal initiation complexes in extracts of Ehrlich ascites tumour cells; this is the stage in initiation previously shown to be regulated in response to lysine starvation of the intact cells. Exposure of cells to a low concentration of cycloheximide makes polypeptide chain elongation the ratelimiting step, allows reformation of polysomes in starved cells and abolishes the effect of lysine deprivation on overall protein synthesis. Treatment with a high concentration of cycloheximide inhibits all protein synthesis and ‘freezes’ the polysomes in the state of aggregation which exists in the cell at that moment. The latter conditions also permit the tRNA pool to become fully charged in both fed and lysine‐starved cells. In cell‐free extracts prepared from cells treated with either low or high concentrations of cyclolieximide 40‐S initiation complex formation nevertheless remains defective after lysine starvation of the cells. Addition of deacylated tRNA, oxidized with periodate to prevent its recharging, to initiating cell‐free extracts inhibits neither 40‐S initiation complex formation nor overall protein synthesis in vitro. Addition of lysine to an extract from lysine‐starved cells, under conditions where this amino acid is charged on to endogenous tRNA, fails to stimulate either 40‐S initiation complex formation or protein synthesis in vitro. These results suggest that amino acid starvation regulates the formation of 40‐S polypeptide chain initiation complexes in mammalian cells by a mechanism which is not influenced by the rate of protein synthesis and does not directly involve uncharged tRNA.

Purine analogs revisited: Interference in protein formation

We have examined in L1210 cells in vitro the effects of three guanine analogs (8-azaguanine, 3-deazaguanine, and 6-thioguanine) on protein biosynthesis and have compared this action with respect to effects on macromolecular synthesis and mechanisms of tumor growth inhibition and cell viability. The major site of action of azaG was the inhibition of protein synthesis. TG seemed to inhibit RNA synthesis more than DNA and protein synthesis. In contrast, DG inhibited both protein and DNA synthesis but not RNA synthesis. Whereas the LD 50 and ID 50 for macromolecular synthesis for DG and also for azaG were quite similar, for TG the LD 50 was one-tenth that of the ID 50 for macromolecular synthesis. Since azaG, DG, and TG had different effects on protein and RNA synthesis, the effects of the analogs on the process of translation were examined. DG and azaG, but not TG, altered the polyribosome sedimentation profile, increasing the numbers of monosomes and smaller polysomes and decreasing the number of larger polysomes. This shift in the polysome profile was reversed by low concentrations of cycloheximide, suggesting that DG and azaG inhibited the initiation of translation. This was examined directly by the incorporation of 35S-met-tRNA f into the initiation complexes. DG and azaG inhibited the formation of the 43S and 80S initiation complexes and this inhibition correlated closely with the inhibition of total protein synthesis. It is likely that the inhibition of tumor growth by azaG is due to the inhibition of initiation of translation, perhaps through actions on mRNA. The mechanism of growth inhibition for DG is not yet known but may similarly involve actions at this level.

Early grey crescent formation experimentally induced by cycloheximide in the axolotl oocyte.

The effects of cycloheximide (CH) on grey crescent formation in artificially maturedAmbystoma mexicanum oocytes were determined. CH induced grey crescent formation after a few hours, especially after a 45° to 90° rotation from the vertical animal-vegetal axis. With low concentrations of CH (about 0.5 ng/oocyte), meiosis was still able to proceed normally to the stable second metaphase stage, but higher concentrations blocked it after 1st polar body extrusion and an interphasic nucleus appeared. Such effects were compared to those of inactone, an analogue of cycloheximide, which as a pure substance does not inhibit protein synthesis, but still contained a small amount of CH in the available samples. It is concluded that grey crescent formation can occur in non-activated oocytes. The effects of cycloheximide might be due to partial inhibition of protein synthesis and the presence of a proteinic inhibitor of the symmetry reaction in the normal oocyte is suggested.

New medium for differentiation of Cryptococcus neoformans serotype pairs.

A new medium effectively differentiates B and C serotypes of Cryptococcus neoformans from A and D serotypes on the basis of resistance to low concentrations of cycloheximide and assimilation of glycine as the sole carbon source.

Changes in corticotropic responsiveness and mitochondrial ultrastructure of adrenocortical cells from the inner zone of the duck (Anas platyrhynchos) adrenal gland: the effects of cycloheximide, puromycin, and chloramphenicol.

Tissue slices from the inner zone of the adrenal showed no ultrastructural abnormalities after being superfused with Krebs-Henseleit buffer for more than 2 h. Slices superfused with medium containing no ACTH produced only traces of corticosterone and most of the mitochondria had shelf-like cristae. Exposure to medium containing ACTH (1 μg 1-24 ACTH · ml-1) caused a significant increase in the release of corticosterone and the mitochondria developed tubular cristae. Cycloheximide and puromycin inhibited, in a dose-dependent manner, the release of corticosterone from tissue slices superfused with medium containing ACTH. A low concentration of cycloheximide in the medium (0.02 μg · ml-1) had only a slight effect on hormone release but many of the mitochondria developed dark matrixes and shelf-like or disrupted cristae. At higher concentrations of cycloheximide (0.2 μg · ml-1) the release of corticosterone was suppressed and when the concentration was increased to 2.0 μg · ml-1 it was blocked completely; each of these higher concentrations of cycloheximide caused the mitochondria to develop dense matrixes and shelf-like cristae. The inhibitory effects of cycloheximide on the corticotropic responsiveness of superfused tissue slices were reversible. Dependent upon the concentration, the addition of puromycin to the medium also caused, either partial (1.0 and 10.0 μm · ml-1) or complete (100 μg · ml-1), suppression of hormone release from slices superfused with medium containing ACTH; at the same time, the formation of tubular cristae in the mitochondria was blocked. Chloramphenicol had no effect on either hormone release or mitochondrial ultrastructure in the adrenocortical cells from the inner zone of the gland. It is suggested that a specific protein, synthesized in the cytoplasm, is necessary to accomplish the conformational changes in the mitochondrial cristae that accompany the full elaboration of corticotropin-induced hormone synthesis.

Induction of Choline Transport and Its Role in the Stimulation of the Incorporation of Choline into Phosphatidylcholine by Polyamines in a Polyamine Auxotroph of Saccharomyces cerevisiae

1. A mutant of Saccharomyces cerevisiae, defective in ornithine decarboxylase, was isolated. A prolonged culture of the mutant in a polyamine-free medium resulted in a great decrease in the polyamine content and in cessation of growth. The addition of polyamines to the culture induced the growth after a lag period of 5--6.5 h. The growth rate in the presence of polyamine was comparable to that of the wild-type strain. The effectiveness of polyamines was as follows: spermidine greater than putrescine approximately equal to spermine. 2. Phosphatidylcholine-synthesizing activity during the lag phase of growth was determined by measuring the rate of incorporation of [14C]choline into phosphatidylcholine. The incorporation rate was markedly increased with time by polyamine prior to the initiation of cell division. Polyamines were effective in the following order: spermidine greater than putrescine approximately equal to spermine. Experiments with methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, showed that putrescine stimulates cell growth and choline incorporation into phosphatidylcholine after it has been converted into spermidine in the cell. 3. The induction of the choline transport system was shown to be responsible for the increase in the rate of incorporation of [14C]choline into phosphatidylcholine effected by polyamines. A low concentration of cycloheximide completely prevented the induction of choline transport by polyamines. The levels of the CDP-choline pathway enzymes such as choline kinase, cholinephosphate cytidyltransferase and cholinephosphotransferase were not significantly changed.

Induction of precocious melanogenesis of pigment cells in cultures of neuroretinal cells of chick embryo by amphotericin B.

When dissociated neuroretinal cells of the 9-day-old chick embryo were cultured, the cells formed monolayer sheets of somewhat flattened epithelial cells within 15 days after inoculation. During 15 to 30 days, numerous foci of non-pigmented epithelial cells were formed. During 30 to 50 days, melanin appeared in the cells of these foci. When amphotericin B (1 microgram/ml) was added to the culture medium on day 25 of culture, brown pigments appeared precociously, i.e. within the first two days, in the cells. The brown pigments were identified as melanins by histochemical and electron-microscopic methods. Induction of melanogenesis required continuous treatment with amphotericin B. With the precocious appearance of melanins, tyrosinase activity increased rapidly. This rapid increase in tyrosinase activity was inhibited by the addition of phenylthiourea or diethyl-dithiocarbamate. It was not enhanced by iodoacetamide, but was blocked by a low concentration of cycloheximide or actinomycin D. These findings indicate that amphotericin B induces de novo synthesis of tyrosinase rather than activation of pre-existing tyrosinase.

Cycloheximide effect on DNA degradation and δ-crystallin synthesis in terminally differentiating lens cells

Low concentrations of a protein synthesis inhibitor, cycloheximide, were added throughout the process of in vitro differentiation of 11-day old embryonic chick lens cells. We found with low concentrations of cycloheximide (0.01 to 0.03 μg/ml, during 3 days of culture), that there was an almost complete delay of DNA degradation as observed on alkaline sucrose gradient. Identical concentrations and exposure time had no blocking effect on increased δ-crystallin synthesis as detected by immunoprecipitation and electrophoresis. Higher concentrations of cycloheximide (0.1 to 1 μg/ml) showed a marked effect on DNA size and a net inhibition on δ-crystallin synthesis. Thus a selective effect of low doses of cycloheximide was observed on terminal differentiation suggesting that there was not a relationship between DNA degradation and δ-crystallin synthesis in these short term experiments. The investigations of minor proteins could be of interest as they may have a crucial role in intact nuclei cataracts.

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.

Studies on the relationship between 5′ leader sequences and initiation of translation of adenovirus 2 and simian virus 40 late mRNAs

The question of whether or not 5′ leader sequences might confer characteristic translational properties to the mRNAs to which they are spliced was studied using two complementary experimental situations with respect to the composition of mRNA molecules. Late in adenovirus type 2 (Ad-2) infection of human cells, a common tripartite 5′ leader sequence is spliced to mRNA sequences coding for at least 13 different late viral proteins. This results in different mRNAs with identical 5′ leader sequences. In the second experimental system, identical coding sequences are spliced to different 5′ leader sequences with different caps. Wild-type SV40 VP1 mRNA (16 S) contains a 5′ leader sequence coded for at position 72–76 on the SV40 genome; in the SV40 mutant dl-808 part of the sequences in this region, including the sequence coding for cap, is deleted. The relative ability of these mRNAs to direct incorporation of radioactive amino acids into their respective polypeptide products was assayed in vivo under conditions of hypertonic initiation block (HIB) or in the presence of low concentrations of cycloheximide. Messenger RNAs with identical 5′ leader sequences were observed to exhibit very different responses in both experimental situations. Conversely, similar responses to HIB were observed for VP1 mRNAs with different 5′ leader sequences. It thus appears that the presence of specific sequences per se between the 5′ cap and the initiation codon does not alone confer characteristic initiation properties as measured indirectly by perturbation of the in vivo environment.

Secretion of lysosomal hydrolases by stimulated and nonstimulated macrophages.

Peritoneal macrophages were obtained from untreated mice and from mice treated with thioglycollate medium (TA), proteose peptone medium (PP), or a suspension of streptococcus A cell wall material (SA). The biochemical and secretory properties of these cells in long term cultures (up to 2 wk) were compared. TA-elicited macrophages contained more protein, lactate dehydrogenase, lysosomal hydrolases, and in particular, more plasminogen activator than the other cells studied. All types of macrophages studied were found to release considerable amounts of lysosomal hydrolases (beta-glucuronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and acid phosphatase) into the medium. Release was independent of phagocytosis and must, therefore, be regarded as true secretion. In both elicited and nonelicited macrophages, the rates of lysosomal enzyme secretion were virtually identical in the presence and in the absence of serum, and they were not enhanced by increasing serum concentrations. Lysosomal enzyme secretion in macrophages appears to depend on protein synthesis, since it was blocked by low concentrations of cycloheximide which neither affected cell viability nor lowered the intracellular enzyme levels. The amounts of lysosomal hydrolases secreted were highest in TA-elicited macrophages. The rates of secretion of PP- or SA-elicited and of nonelicited macrophages were about one-fourth of that of the TA-elicited cells. This difference, although significant, is much smaller than that observed for the secretion of plasminogen activator which was 20-50 times higher in TA-elicited cells. Acid glycosidases were also found in the peritoneal lavage media used for cell harvesting from both treated and nontreated mice. This indicates that active secretion of lysosomal hydrolases may be an in vivo property of the macrophage.

Effect of cycloheximide on maturation of replicative intermediates into high-molecular-weight DNA in Tetrahymena.

Replicative intermediates of discrete size (approximately 41 S) are observed in the eukaryotic organism Tetrahymena pyriformis, when the organism is grown under defined physiological conditions. The intermediates (believed to represent replicons) are synthesized and accumulated over longer periods of time (less than 90 min), if the cells are treated with low concentrations of cycloheximide. Under these conditions the rate of total DNA synthesis is only slightly inhibited (less than 15%), while maturation of intermediate DNA into high-molecular-weight DNA is completely blocked (greater than 98%). Cycloheximide appears to inhibit the maturation process more specifically than other protein synthesis inhibitors. Studies of the accumulated intermediates on alkaline buoyant density gradients demonstrate that initiation of new putative replicons occurs during treatment with cycloheximide.

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases.

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyl-transfer RNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 microgram/ml), all three log phase enzymes showed an increase in activity of 40-50%. At slightly higher concentrations (2.5 microgram/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5 microgram/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5-20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5 microgram/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10 microgram/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.