Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

Control of sterol metabolism in rat adrenal mitochondria

Steroidogenesis by adrenal mitochondria from endogenous precursors is stimulated by corticotropin (ACTH) and is sensitive to the protein-synthesis inhibitor cycloheximide. In the present investigation the effect of cycloheximide treatment on the metabolism of a number of analogues of the normal steroidogenic substrate, i.e. cholesterol, by rat adrenal mitochondria was studied. It was observed that the metabolism of analogues such as desmosterol, 26-norcholest-5-en-3β-ol and 5-cholen-3β-ol (that is with non-polar alkyl side chains like cholesterol), was sensitive to cycloheximide treatment. By contrast, the metabolism of those analogues with polar groupings on the side chain, i.e., 20α-, 24-, 25- and 26-hydroxycholesterols was insensitive to pretreatment with cycloheximide. The binding of added sterol to the cytochrome P-450 component of the mitochondrial sterol desmolase was studied. Similar studies on the equilibration time on addition of exogenous sterols to achieve maximum rates of pregnenolone production were also made. Both studies show that cholesterol, a non-polar sterol, penetrated slowly through the mitochondrial milieu to reach the cytochrome P-450 reaction centre whereas 24- and 26-hydroxycholesterols rapidly attained the enzymic environment. The cycloheximide-sensitive process in sterol metabolism appeared related to the transfer of non-polar sterols such as cholesterol within the mitochondria to a region in close proximity to the enzyme. The importance, and possible mechanism of action, of the cycloheximide-sensitive factor in the control of adrenal steroidogenesis is discussed.     

Inactivation of 1,6-Diphosphatase by Glucose in Yeast

Fructose-1,6-diphosphatase was derepressed in Saccharomyces cerevisiae by incubation in media containing non-sugar carbon sources. Addition of glucose to a derepressed culture led to a rapid loss of the measurable activity of the enzyme. Fructose and mannose also produced inactivation, but 2-deoxyglucose was ineffective. Experiments with cycloheximide indicated that the inactivation does not require protein synthesis. It was also shown that the process is not energy-dependent. The reappearance of the enzyme was dependent on an energy source and was prevented by cycloheximide. These results suggest that fructose diphosphatase inactivation is irreversible and that reappearance of enzyme activity implies de novo synthesis. Screening of different genera of yeasts has shown that the inactivation of fructose diphosphatase is a relatively widespread phenomenon.     

Synchronous Cultures of Chlamydomonas reinhardti: Properties and Regulation of Repressible Phosphatases

De novo synthesis of phosphatase (derepression) in orthophosphate deprived synchronously growing Chlamydomonas reinhardti has been demonstrated by using a double labelling isotope technique coupled with cellulose acetate electrophoresis. Repressed and derepressed phosphatase exhibited different enzymatic properties as pH optimum, electrophoretic pattern, K_m and K_i values. Especially the acid phosphatase was located near the cell surface. Inorganic, cold TCA-extractable ³²P, decreased during the first 1–2 h after phosphate deprivation when there was little or no net synthesis of phosphatase. Results of experiments with additions of orthophosphate and cycloheximide to derepressed cells, indicated that the derepressible enzyme was relatively unstable, while its m-RNA was relatively stable.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.     

Phosphate transport in Neurospora. Derepression of a high-affinity transport system during phosphorus starvation

In addition to the constitutive, low-affinity phosphate-transport system described previously, Neurospora possesses a second, high-affinity system which is derepressed during phosphorus starvation. At pH 5.8, System II has a of about 3μM and a J_max of 5.2 mmol/l cell water per min.System II reaches maximal activity after about 2 h of growth in phosphorus-free minimal medium. Its formation is blocked by cycloheximide and, once made, it appears to turn over rapidly. Addition of cycloheximide to fully derepressed cultures results in the decay of System II with a of 14 min, very similar to the turnover rate previously reported (Wiley, W.R. and Matchett, W.H. (1968) J. Bacteriol. 95, 959–966) for tryptophan transport in Neurospora. Thus, these transport systems appear to be regulated by a balance between synthesis and breakdoown, as affected by intracellular pools of substrate or related compounds.     

The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase

Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6-9.5).     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes.

Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.     

Continuous synthesis of partially derepressed aspartate transcarbamylase during the division cycle of Escherichia coli B-r

The pattern of synthesis of aspartate transcarbamylase during the division cycle of Escherichia coli B/r was determined for several steady-state levels of partially derepressed synthesis of the enzyme. The pattern of synthesis was measured by repressing ATCase synthesis with uracil in populations growing on the surfaces of membrane filters and measuring enzyme activity as a function of time in newborn cells eluted from the populations. The variations in levels of steady-state partial derepression of ATCase synthesis were achieved by including 6-aza-uracil in the growth medium or by using a mutant possessing an increased level of partially derepressed ATCase synthesis compared to the parental strain. The results indicated that the enzyme was synthesized continuously during the division cycle at all levels of partial derepression. The observed absence of step-wise ATCase synthesis during the division cycle, which had been reported previously, was not due to a limitation of the method of analysis since a step-wise pattern was observed when it was expected, i.e. after ATCase synthesis was pulse-derepressed in an exponentially growing population. The possibility that previous reports of periodic synthesis of partially derepressed ATCase in E. coli (Kuempel et al., 1965; Goodwin, 1969a) were manifestations of a disturbance in cellular metabolism caused by the techniques used for synchronization is discussed.     

The regulation of the decay of nitrate reductase Evidence for the existence of at least two mechanisms of decay

There seem to be at least two different mechanisms of decay of nitrate reductase in Neurospora in vivo: one which is very sensitive to EDTA and cycloheximide, decreases with mycelial age and is not increased by an increase in temperature from 27 to 37°C, the other which is relatively insensitive to EDTA and cycloheximide, increases with the age of the mycelium and with the above temperature shift.     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Phenylalanine Biosynthesis in Escherichia coli K-12: Mutants Derepressed for Chorismate Mutase P-Prephenate Dehydratase

Mutants were isolated which are derepressed for the synthesis of chorismate mutase P-prephenate dehydratase. No other enzymes involved in the synthesis of phenylalanine are derepressed in these strains. These mutants are able to grow in concentrations of o- and p-fluorophenylalanine that inhibit the growth of AB3259, the strain from which they were derived. They also excrete phenylalanine. Genetic analysis shows that the mutations causing this derepression are closely linked to the structural gene for this enzyme (cotransduction frequency of 95% or more with pheA). The gene in which they occur has been designated pheO since this gene has all of the properties predicted for an operator gene controlling the pheA structural gene. Finally, the pheO mutant alleles have been shown to be dominant in diploids.     

Regulation of choline sulphatase synthesis and activity in Aspergillus nidulans

1. Choline O-sulphate is taken up from the growth medium to the same extent by sulphur-deficient and sulphur-sufficient mycelia of Aspergillus nidulans, but hydrolysis of the transported sulphate ester in vivo only occurs in the sulphur-deficient mycelia. 2. Choline sulphatase activity could not be detected in vitro in sulphur-sufficient mycelia of wild-type and sulphur mutants of A. nidulans, but after sulphur starvation all strains showed appreciable activity of this enzyme. 3. Optimum activity of choline sulphatase in an ultrasonically treated preparation of sulphur-deficient mycelia was at pH7.5. The optimum substrate concentration was in excess of 25mm and K(m) was 0.035m. The enzyme was completely inhibited by 10mm-SO(3) (2-), PO(4) (3-), CN(-) and cysteine. 4. Growth of sulphur-deficient mycelia on various sulphur sources resulted in a decrease of choline sulphatase activity in vitro. The decrease appeared to be due to a repression of choline sulphatase synthesis rather than to inhibition of activity. De-repression by growth on a sulphur-deficient medium was prevented by cycloheximide. Unlike the choline sulphatase of bacteria the fungal enzyme did not need to be substrate-induced. 5. By using sulphur mutants the identity of the co-repressor was limited to S(2)O(3) (2-), cysteine-S-sulphonate, cysteine or compounds derived directly from them. Circumstantial evidence suggests that the co-repressor is cysteine. 6. Inhibition of choline sulphatase activity in vivo was demonstrated with cysteine as the sulphur source for growth.     

Rapid activation by phytochrome of nitrate reductase in the cotyledons of Sinapis alba

Summary Nitrate reductase in the cotyledons of etiolated seedlings of Sinapis alba L. responds rapidly to the addition of nitrate. The response is inhibited by cycloheximide at low concentrations. The enzyme is also under phytochrome control. Five minutes of red light irradiation leads instantaneously to a 45% increase in enzyme activity. Increases in activity, linear with respect to time and with no lag phases are promoted by continuous far-red or blue irradiation. These increases are insensitive to cycloheximide. Thus, light and nitrate act through different mechanisms in controlling nitrate reductase activity and phytochrome does not act via controlling the rate of synthesis of the enzyme.Abbreviation cot pr pair of cotyledons     

Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. Production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. Isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. The lah-1 mutation is mapped between nit-2 and leu-3 on linkage group I, and it behaved as a recessive mutation in a forced heterokaryon. No differences were detected biochemically or immunologically between the laccase protein produced by the lah-1 mutant in the absence of cycloheximide and that induced with cycloheximide in the wild-type strain. This suggests that both laccases (66 kilodaltons) are products of the same structural gene. Relative amounts of laccase in the culture filtrate of the lah-1 mutant were much higher than those induced with cycloheximide in the wild-type strain, demonstrating high efficiency of the lah-1 mutant in production and secretion of laccase. The time course of laccase production by the lah-1 mutant revealed that expression of 66-kilodalton laccase was repressed in conidia and derepressed during vegetative mycelial growth. This suggests that a multiple regulatory mechanism is involved in the production and/or maturation of Neurospora laccase. The lah-1 mutant may be useful for identifying genes that regulate expression of the laccase gene in N. crassa.     

Regulation of fructose 2,6-bisphosphate metabolism in human fibroblasts

In the present work, the mechanism involved in the regulation of fructose 2,6-bisphosphate (fructose-2,6-P₂) metabolism in human fibroblasts has been studied. Various agents like serum, insulin and adrenaline known to affect glycolysis have been investigated for their ability to influence fructose 2,6-P₂ metabolism in confluent human fibroblasts. Serum appears to be the most potent activator of fructose-2,6-P₂ levels and capable of inducing a marked increase in 6-phosphofructo-2-kinase (ATP: d-fructose-6-phosphate-2-phosphotransferase), EC 2.7.1. 105). To a lesser extent insulin has the same effects. The increase in enzyme activity elicited by serum and insulin does not require de novo protein synthesis since the process is insensitive to cycloheximide. Incubation of fibroblasts in the presence of adrenaline is responsible for a significant rise in fructose-2,6-P₂ levels without affecting 6-phosphofructo-2-kinase. Similar experiments performed on glucose-starved or cytochalasin B-treated cells show that the effects elicited by all the agents are strictly dependent on glucose availability.     

Mechanism of choline O-sulphate utilization in fungi

1. The position of the enzyme blocks in a number of parathiotrophic mutants of Aspergillus nidulans A 69 and mutants A and C of a biotinless mutant of Aspergillus nidulans were examined by nutritional and heterokaryosis experiments and by assay in vitro of enzyme systems and specific enzymes. 2. The mutants were in five groups: A and C blocked at sulphate transport; gamma at ATP sulphurylase; iota at adenosine 5′-sulphatophosphate kinase; eta at the adenosine 3′-phosphate 5′-sulphatophosphate reductase system; alpha, beta and zeta between sulphite and thiosulphate. 3. The ability of the various mutants to synthesize choline O-sulphate in vivo and in vitro and to utilize choline O-sulphate as a source of sulphur indicated that the utilization of endogenously formed choline O-sulphate involved the splitting off of inorganic sulphate, which was then reduced. 4. Choline O-sulphate acted as a source of choline for cholineless strains of Neurospora crassa, suggesting that choline O-sulphate breakdown occurred by simple hydrolysis involving a choline sulphatase. 5. After de-repression of mycelia by growing for a period on a sulphur-free medium the presence of choline sulphatase in physiologically significant amounts was demonstrated in all the A. nidulans strains tested. 6. Choline O-sulphate is transported across the mycelial wall by a mechanism different to that responsible for inorganic sulphate transport.