Cytosolic Protein Synthesis Research Articles

The Light-Dependent Transduction Pathway Controlling the Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase in Protoplasts from Digitaria sanguinalis.

Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent protein-serine kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent protein kinase and a cytosolic protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.

Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins

Previously we have found that stationary Ehrlich ascites carcinoma (EAC) cells in vivo accumulated heat shock proteins (HSPs) and became resistant to necrotic heath induced by prolonged energy deprivation of hyperthermia. Here we report that apoptotic death induced by nutrient starvation, transient ATP depletion, heat shock and a microtubule-disrupting drug, vinblastine, was also suppressed in stationary EAC cells comparing with exponential cells. When exponential (sensitive) cells were subjected to short-term heating with recovery to accumulate inducible form of HSP70, they also became resistant to all of the employed apoptosis-inducing exposures, and an inhibitor of cytosolic protein synthesis, cycloheximide, prevented acquisition of the resistance. It is suggested that in vivo accumulation of HSPs in stationary tumor cells can be endogenous protective device against apoptotic death induced by starvation or some anticancer treatments.

Mode of action of the anilino‐pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea

AbstractThe effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liquid B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5.0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day‐old cultures grown in the presence of pyrimethanil. The pyrimethanil concentrations resulting in 50% reduction in total enzyme activities (IC50) were approximately 0.25 μM for polygalacturonase, cellulase and proteinase, and approximately 1.0 μM for laccase. No significant growth inhibition was observed at these pyrimethanil concentrations. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post‐translational stage in the secretory pathway.Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liquid cultures and on agar plates, depending on the composition of the medium. In liquid media containing cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5.0 μM pyrimethanil, whilst no growth inhibition was observed with 50 μM pyrimethanil in malt extract. Similarly, growth occurred on potato/dextrose agar (PDA) at 0.5 μM pyrimethanil, but no growth was seen at this concentration on agars containing cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilise extracellular enzymes to mobilise the nutrients it requires for growth.

The Relative Importance of Mitochondrial Protein Synthesis to Brewing Yeast Performance

Using the inhibitors chloramphenicol and cycloheximide, the relative importance of mitochondrial vs. cytosolic protein synthesis to brewing yeast performance and final beer quality was assessed. Cytosolic protein synthesis was much more critical, but mitochondrial protein synthesis did affect the overall performance of a commercial lager strain of Saccharomyces cerevisiae in high-gravity (16° Plato) brewers' wort. Previous research had indicated a critical role for mitochondrial energy in the promotion of adequate fermentation despite the fact that brewing yeasts reportedly obtained all of their energy from fermentative growth. This research indicated that mitochondrial protein synthesis was involved in yeast growth, specific carbohydrate uptake rates, free amino nitrogen utilization, ethanol production, and lipid and glycogen biosynthesis. This was true for yeast in two different physiological conditions used for inoculation. Artificially aged yeast showed a greater susceptibility to the inhibitors than did brewery-fresh yeast, presumably due to permeability differences and/or a lack of ability for the compromised cells to counteract the inhibitor damage. The blockage of mitochondrial protein synthesis did not alter the yeast performance as radically as did inhibiting the energy supply to these organelles but did inhibit the commercial yeast strain's ability to ferment optimally. It is suggested that mitochondrial protein synthesis occurs during the brewing fermentation and contributes to the efficiency and end quality of the product and the yeast harvested from the fermentation for reuse. However, protein synthesis in the mitochondria was not the major contributor to previous observations, indicating a critical role for mitochondria in brewing yeast performance.

The Relative Importance of Mitochondrial Protein Synthesis to Brewing Yeast Performance

Using the inhibitors chloramphenicol and cycloheximide, the relative importance of mitochondrial vs. cytosolic protein synthesis to brewing yeast performance and final beer quality was assessed. Cytosolic protein synthesis was much more critical, but mitochondrial protein synthesis did affect the overall performance of a commercial lager strain of Saccharomyces cerevisiae in high-gravity (16° Plato) brewers' wort. Previous research had indicated a critical role for mitochondrial energy in the promotion of adequate fermentation despite the fact that brewing yeasts reportedly obtained all of their energy from fermentative growth. This research indicated that mitochondrial protein synthesis was involved in yeast growth, specific carbohydrate uptake rates, free amino nitrogen utilization, ethanol production, and lipid and glycogen biosynthesis. This was true for yeast in two different physiological conditions used for inoculation. Artificially aged yeast showed a greater susceptibility to the inhibitors than did brewery-fresh yeast, presumably due to permeability differences and/or a lack of ability for the compromised cells to counteract the inhibitor damage. The blockage of mitochondrial protein synthesis did not alter the yeast performance as radically as did inhibiting the energy supply to these organelles but did inhibit the commercial yeast strain's ability to ferment optimally. It is suggested that mitochondrial protein synthesis occurs during the brewing fermentation and contributes to the efficiency and end quality of the product and the yeast harvested from the fermentation for reuse. However, protein synthesis in the mitochondria was not the major contributor to previous observations, indicating a critical role for mitochondria in brewing yeast performance.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

Protein turnover is generally regarded as a major maintenance process, but experimental evidence to support this contention is scarce. Here we quantify the component of dark respiration rate associated with overall protein turnover of tissues in vivo. The effect of an inhibitor of cytosolic protein synthesis (cycloheximide, CHM) on dark respiration was tested on a cell suspension from potato (Solanum tuberosum L.) and quantified on leaf discs of expanding and full‐grown primary leaves of bean (Phaseolus vulgaris L.). The in vivo effect of CHM on protein biosynthesis was assessed by monitoring the inhibition of the induction of the ethylene‐forming enzyme (EFE) activity. The present method yields the energy costs of turnover of the total pool of proteins irrespective of their individual turnover rates. Average turnover rates were derived from the respiratory costs and the specific costs for turnover.Inhibition of respiration by CHM was readily detectable in growing‐cell suspensions and discs of expanding leaves, The derived respiratory costs of protein turnover in expanding leaves were maximally 17–37% of total respiration. Turnover costs in full‐grown primary leaves of bean amounted to 17–21% of total dark respiration. The maximum degradation constants (i.e. Kd‐values) derived for growing and full‐grown leaves were up to 2.42 × 10−6 and 1.12 × l0−6 s−1, respectively.

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by atp

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.

Rise in heat-shock protein level confers tolerance to energy deprivation

Heat shock (44°C for 10 min) or ATP depletion by an uncoupler (CCCP for 20 min) is shown to result in stimulation of hsp68 70 synthesis in Ehrlich tumor cells. After 3 h of recovery, the cells become thermotolerant. Surprisingly, repeated ATP depletion caused by CCCP or rotenone (a respiratory inhibitor) treatment, had a much lower effect on cell viability. Both induction of tolerance to energy deprivation and hsp68 70 synthesis were totally suppressed by cycloheximide, an inhibitor of cytosolic protein synthesis. In tolerant cells, rotenone still induced ATP depletion; however, protein aggregation (the rise in Triton-insoluble proteins) was inhibited in these cells. It is suggested that cellular chaperones (e.g. hsp70) are involved in the protection of ischemie cells from necrosis, preventing protein aggregation under ATP deficiency.

Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo.

We have utilized a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae, in addition to performing a series of in vivo experiments in yeast, to investigate the coupling between cytosolic protein synthesis and protein transport into mitochondria. We found that the import of bulk mitochondrial proteins was inhibited in both the homologous in vitro reaction and in vivo upon arrest of cytosolic protein synthesis with the addition of cycloheximide. Tight coupling of synthesis and import was also demonstrated in vivo for the beta subunit of the mitochondrial F1-ATPase. We also investigated the effect of the antifolate methotrexate on the import of a fusion protein consisting of the mitochondrial targeting signal of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (the COXIV-DHFR fusion protein). Methotrexate has previously been shown to inhibit posttranslational import of COXIV-DHFR by preventing the DHFR moiety from unfolding. However, we found that antifolate addition had no inhibitory effect on the import of COXIV-DHFR in vivo, suggesting that its import into mitochondria in yeast cells occurs cotranslationally. Further, when we treated yeast with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone to collapse the mitochondrial membrane potential and induce the accumulation of extramitochondrial precursor pools, we found that the ability to be imported by a strictly posttranslational mechanism upon reestablishing the membrane potential varied from one precursor to another, suggesting that cotranslational import may be mandatory for the import of some proteins in vivo. In summary, our findings are entirely consistent with the notion that import of proteins into yeast mitochondria occurs cotranslationally under normal conditions in vivo.

Regulatory phosphorylation of phosphoenolpyruvate carboxylase in protoplasts from Sorghum mesophyll cells and the role of pH and Ca2+ as possible components of the light-transduction pathway.

The light-dependent phosphorylation of the photosynthetic phosphoenolpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. It was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i.e. an increase in Vmax and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4Cl and methylamine (agents which increase cytosolic pH), but not by KNO3, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca(2+)-selective ionophore A23187 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2+, to the Ca(2+)-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activity/PyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.

Regulatory phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase. Identification of the protein-serine kinase and some elements of the signal-transduction cascade.

The phosphoenolpyruvate (PPrv) carboxylase isozyme involved in C4 photosynthesis undergoes a day/night reversible phosphorylation process in leaves of the C4 plant, Sorghum. Ser8 of the target enzyme oscillates between a high (light) and a low (dark) phosphorylation status. Both in vivo and in vitro, phosphorylation of dark-form carboxylase was accompanied by an increase in the apparent Ki of the feedback inhibitor L-malate and an increase in Vmax. Feeding detached leaves various photosynthetic inhibitors, i.e. 3-(3,4-dichlorophenyl)-1,1-dimethylurea, gramicidin and DL-glyceraldehyde, prevented PPrv carboxylase phosphorylation in the light, thus suggesting that the cascade involves the photosynthetic apparatus as the light signal receptor, and presumably has the electron transfer chain and the Calvin-Benson cycle as components in the signal-transduction chain. Two protein-serine kinases capable of phosphorylating PPrv carboxylase in vitro have been partially purified from light-adapted leaves. One was isolated on a calmodulin-Sepharose column; it was calcium-dependent but did not require calmodulin for activity. The other was purified on a blue-dextran-agarose column and the only Me2+ required for activity was Mg2+. In reconstituted phosphorylation assays, only the latter caused the expected decrease in malate sensitivity of PPrv carboxylase suggesting that this protein is the genuine PPrv-carboxylase-kinase. Desalted extracts from light-adapted leaves possessed a considerably greater phosphorylation capacity with immunopurified dephosphorylated PPrv carboxylase as substrate than did dark extracts. This light stimulation was insensitive to type 2A protein phosphatase inhibitors, okadaic acid and microcystin-LR, which suggests that the kinase is a controlled step in the cascade which leads to phosphorylation of PPrv carboxylase. The higher phosphorylation capacity of light-adapted leaf tissue was nullified by pretreatment with the cytosolic protein synthesis inhibitor, cycloheximide. Thus, protein turnover is involved as part of the mechanism controlling the activity of the kinase purified on blue-dextran-agarose. However, no information is available with respect to the specific nature of the link between the above-mentioned light transducing steps and the protein kinase that achieves the physiological response. Finally, the in vivo phosphorylation site (Ser8) in the N-terminal region of the C4 type Sorghum PPrv carboxylase is also present in a non-photosynthetic form of the Sorghum enzyme (Ser7), as deduced by cDNA sequence analysis.

Transport of l-glutamic acid in the fission yeast Schizosaccharomyces pombe

Transport of l-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% d-glucose is practically unidirectiional and is mediated by a single uphill transport system with a K T of 170 μM and J max of 4.8 nmol min −1 (mg dry wt.) −1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0–4.0, the accumulation ratio of l-glutamic acid is highest at a suspension density of 0.6–1.0 mg dry wt. per ml and decreases with increasing l-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with d-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.

Qualitative and quantitative changes in protein sythesis of bovine follicular cells during the preovulatory period

To understand the mechanisms governing oocyte maturation better, the effects of the gonadotropin surge were studied on follicular cells of bovine preovulatory follicles. For this purpose, qualitative and quantitative changes in protein synthesis by both granulosa cells and cumulus cells were compared relative to the luteinizing hormone (LH) surge and the resumption of meiosis in the oocyte. Follicular cells were collected at different times before and up to 25 hr after the LH surge. For each individual preovulatory follicle, granulosa and cumulus cells were incubated separately for 3 hr with 3H-methionine or with 35S-methionine. Newly synthesized cytosolic proteins from granulosa and cumulus cells and proteins secreted into the medium were analyzed by polyacrylamide gel electrophoresis. The radioactivity was measured by liquid scintillation counting after slicing of the gels or revealed by fluorography. Three major peaks of the newly synthesized proteins, with molecular weights of 76, 56, and 30 kDa, were studied throughout the preovulatory period. After the LH surge, the overall level of protein synthesis increased in granulosa cells. In addition, the pattern of cytosolic proteins in granulosa cells changed, and, in particular, the relative synthesis of the 30 kDa peak decreased. These changes in cytosolic protein synthesis may be due to the action of LH since they could be reproduced in vitro in LH-stimulated granulosa cells. A predominant peak of 56 kDa was secreted by granulosa cells throughout the experimental period. No significant change was observed in proteins synthesized by cumulus cells under the same experimental conditions. The amounts of radioactivity incorporated into the three major proteins secreted by granulosa cells, however, were correlated significantly with the amounts of radioactivity incorporated by similar proteins synthesized by cumulus cells. These results indicate that cumulus cells respond differently from granulosa cells to the gonadotropin surge but not in an independent manner.

The Neurospora crassa cyt-20 Gene Encodes Cytosolic and Mitochondrial Valyl-tRNA Synthetases and May Have a Second Function in Addition to Protein Synthesis

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.

The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.

Water stress, carbon dioxide, and light effects on sucrose-phosphate synthase activity in Phaseolus vulgaris

The characteristics of sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various CO2 and light treatments. When water was withheld for 3 days causing mild water stress (–0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6-phosphate and UDP glucose. Placing a water-stressed plant in an atmosphere containing 1% CO2 reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO2 partial pressure reduced the extractable activity of SPS. After 1 h of low CO2 treatment the effect of low CO2 could be reversed by 20 min of 5% CO2. However, after 24 h of low CO2 treatment, less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the slow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low CO2 in that the effects were most pronounced under Vmax assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species.

Water stress, carbon dioxide, and light effects on sucrosephosphate synthase activity in Phaseolus vulgaris

The characteristics of sucrose‐phosphate synthase (SPS; EC 2.4.1.14) activity in leaves of Phaseolus vulgaris L. cv. Linden was studied in plants subjected to water stress and various CO2 and light treatments. When water was withheld for 3 days causing mild water stress (–0.9 MPa), the activity of SPS measured in crude extracts was reduced ca 50%. The effect of water stress was most evident when the enzyme was assayed with saturating amounts of its substrates fructose 6‐phosphate and UDP glucose. Placing a water‐stressed plant in an atmosphere containing 1% CO2 reversed the effect of water stress on SPS activity over 5 h even though the water stress was not relieved. Holding unstressed leaves in low CO2 partial pressure reduced the extractable activity of SPS. After 1 h of low CO2 treatment the effect of low CO2 could be reversed by 20 min of 5% CO2. However, after 24 h of low CO2 treatment, less SPS activity was recovered by the 20 min treatment. The cytosolic protein synthesis inhibitor cycloheximide prevented the slow recovery of SPS activity, but did not affect the rapid recovery of SPS. We conclude that the effect of water stress on SPS activity was a consequence of the inhibition of photosynthesis caused by stomatal closure. Responses of Phaseolus vulgaris SPS to light were similar to the response to low CO2 in that the effects were most pronounced under Vmax assay conditions. This is the first report of this type of light response of SPS in a dicotyledonous species.

Breakdown of Chlorophyll in Chloroplasts of Senescent Barley Leaves Depends on ATP

Chloroplasts isolated from senescent mesophyll of barley primary leaves were tested for the production of one of the blue-fluorescent putative catabolites of chlorophyll, FC 2, of which a small pool is present in senescent organelles. When illuminated during incubation, intact chloroplasts produced this compound for up to 1h. The reaction was abolished by DCMU, an inhibitor of photosynthetic electron transport. In darkness, the reaction continued for at least 4h if the chloroplasts were supplemented with ATP. In presenescent chloroplasts the ATP-dependent accumulation of FC 2 occurred at a very low rate. The catalist(s) responsible for the reaction appears to be induced during the main period of chlorophyll breakdown: its activation, in leaves induced to senesce, was prevented by cycloheximide, an inhibitor of cytosolic protein synthesis. The activity described is likely to represent the key reaction in chlorophyll breakdown, namely the conversion of chlorophyllide into a product, FC 2, having an oxidatively opened tetrapyrrol ring system.

Effect of antioxidants on the mitochondrial activity and toxicity of the cancer drug methylglyoxal bis (guanylhydrazone) in yeast and mammalian cells

Mitochondria of yeast cells were primary targets of methylglyoxal bis (guanylhydrazone) (MGBG) from the following criteria: (1) selective inhibition of growth of cells utilizing a nonfermentable energy source, (2) inhibition of mitochondrial protein synthesis compared with cytosolic protein synthesis and (3) selective mutagenesis of the mitochondrial genome compared with nuclear mutagenesis. Evidence of primary antimitochondrial activity of MGBG in mammalian cells was provided by greater potency of the drug in guinea pig keratinocyte cultures utililizing glutamine as carbon and energy source compared with fermentable glucose. Cell death was used as a measure of drug toxicity in both yeast and mammalian systems. The antioxidants glutathione, vitamin E and vitamin C reversed toxicity and antimitochondrial activity to a large extent implying that toxic free radical metabolites of the drug are of significance in cellular activity of MGBG.

Temperature Dependence of Phase Response Curves for Drug-Induced Phase Shifts

The effectiveness of drugs active in phase-shifting the circadian rhythm of bioluminescent glow in the unicellular dinoflagellate Gonyaulax polyedra differs, depending upon the time of drug exposure (as pulses). For two drugs tested--cycloheximide and anisomycin, both inhibitors of cytosolic protein synthesis--this function, referred to as the drug phase response curve (dPRC), differs, depending upon the ambient temperature. Since dPRCs may differ at different drug concentrations, the effects observed may be attributable to differences in the effectiveness of or recovery from the drugs at different temperatures.