Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.     

The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm.

The capacity of castor-bean endosperm tissue to incorporate [35S]methionine into proteins of the total particulate fraction increased during the first 3 days of germination and subsequently declined. At the onset of germination 66% of the incorporated 35S was found in the separated endoplasmic-reticulum fraction, with the remainder in mitochondria, whereas at later developmental stages an increasing proportion of 35S was recovered in glyoxysomes. The kinetics of [35S]methionine incorporation into the major organelle fractions of 3-day-old endosperm tissue showed that the endoplasmic reticulum was immediately labelled, whereas a lag period preceded the labelling of mitochondria and glyoxysomes. When kinetic experiments were interrupted by the addition of an excess of unlabelled methionine, incorporation of [35S]methionine into the endoplasmic reticulum rapidly ceased, but incorporation into mitochondia and glyoxysomes continued for a further 1h. Examination of isolated organelle membranes during this period showed that the addition of unlabelled methionine resulted in a stimulated incorporation of [35S]no methionine into the endoplasmic-reticulum membrane for 30 min, after which time the 35S content of this fraction declined, whereas that of the glyoxysomal membranes continued to increase slowly. The 35S-labelling kinetics of organelles and fractions derived therefrom are discussed in relation to the role of the endoplasmic reticulum in protein synthesis during glyoxysome biogenesis.     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Optimization of in vitro protein synthesis by isolated mouse adrenal mitochondria

The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of protein synthesis in Y-organs of the blue crab (Callinectes sapidus): involvement of cyclic AMP

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-Organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide produced and released by the X-organ/sinus gland complex of the eyestalks. In the present studies, crab (Callinectes sapidus) Y-organs were incubated in vitro in the presence of [(35)S]methionine, and cyclic nucleotide analogs or experimental agents that influence the cAMP signaling pathway. In 4-hr incubations, 8-Br-cAMP and db-cAMP (but not 8-Br-cGMP) suppressed incorporation of [(35)S]methionine into Y-organ proteins; the effect of 8-Br-cAMP was concentration-dependent. Autoradiograms of radiolabeled Y-organ proteins separated on SDS-PAGE gels indicated the effect of 8-Br-cAMP was general (as opposed to selective) suppression of protein synthesis. Addition of both forskolin (an adenylyl cyclase activator) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) likewise suppressed incorporation of [(35)S]methionine into Y-organ proteins. Cycloheximide (a protein synthesis inhibitor) suppressed incorporation of [(35)S]methionine into Y-organ proteins and secretion of ecdysteroids. The combined results suggest that cAMP is involved in regulation of protein synthesis in C. sapidus Y-organs. We are currently investigating the link of protein synthesis to ecdysteroid production, and the possibility of cross-talk between cAMP and other cellular signaling pathways in Y-organs.     

Brugia pahangi: inhibition of protein synthesis

The effects of inhibitors of protein synthesis and electron transport on the incorporation of [14C]leucine and [35S]methionine into protein by the filarial worm Brugia pahangi have been investigated. Cycloheximide inhibits the accumulation of both [14C]leucine and [35S]methionine by the worms and their incorporation into protein. In addition, inhibitors of electron transport and some anti-parasitic compounds also significantly inhibit filarial protein synthesis. Antimycin A and cyanide inhibit [14C]leucine incorporation into protein 63 and 72%, respectively, without affecting either motility or lactate production. Interestingly, the anti-malarial compounds chloroquine and quinacrine also significantly inhibit both accumulation and incorporation of [14C]leucine by B. pahangi. In addition, fluorographs of sodium dodecyl sulfate-polyacrylamide gels of homogenates from filariids incubated in [35S]methionine and cycloheximide with and without chloramphenicol indicate that there is a discrete population of proteins, possibly mitochondrial in origin, that are synthesized in the presence of cycloheximide and are not inhibited by chloramphenicol.     

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.     

Ozone-Induced Alterations in the Accumulation of Newly Synthesized Proteins in Leaves of Maize

We examined the response of leaves of 3-week-old maize (Zea mays L.) to short-term (5 h) fumigation with O3-enriched air (0, 0.12, 0.24, or 0.36 [mu]L/L). Older leaves and leaf tissue developed more severe visible damage at higher external O3 concentrations. To investigate the immediate effect of O3 exposure on the accumulation of newly synthesized leaf proteins, leaves were labeled with [35S]methionine after 2 h and fumigated for an additional 3 h. O3-induced alterations of leaf proteins were observed in a concentration-dependent manner. There was a significant decrease in [35S]methionine incorporation into protein at the highest O3 concentration. Developmental differences in accumulation of de novo-synthesized leaf proteins were observed when the leaf tip, middle, and basal sections were labeled under 0 [mu]L/L O3, and additional changes were apparent upon exposure to increasing O3 concentrations. Changes in leaf protein synthesis were observed in the absence of visible leaf injury. Subcellular fractionation revealed O3-induced alterations in soluble and membrane-associated proteins. A number of thylakoid membrane-associated proteins showed specific increases in response to O3 fumigation. In contrast, the synthesis of a 32-kD polypeptide associated with thylakoid membranes was reduced in response to O3 fumigation in parallel with reduced incorporation of [35S]methionine into protein. Immunoprecipitation identified this polypeptide as the D1 protein of photosystem II. A reduction in the accumulation of newly synthesized D1 could have consequences for the efficiency of photosynthesis and other cellular processes.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Effect of Intravenous Administration of D-Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell-Free System Derived from Brain

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.     

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).     

The effect of Ca2+ ionophores upon the synthesis of proteins in cultured skeletal muscle

The influence of the Ca2+ ionophores, ionomycin and A23187 upon the incorporation of [35S]methionine into proteins of cultured chicken pectoralis muscle was studied during differentiation of myoblasts into multinucleated myotubes. Fusion was reversibly arrested by growing cells in low-calcium media from the time of plating. Exposure of normal and fusion blocked cultures to 10-6-10-5 M ionomycin or A23187 for 2-6 h on the second to fourth day of growth, resulted in a selective increase in the incorporation of [35S]methionine into two proteins of about 100 000 and 80 000 dalton. When 10-5 M ionomycin or A23187 were added to older cultures, all large myotubes contracted and detached from the plate. Only the adhering myoblasts and small myotubes incorporated [35s[methionine into the muscle proteins and showed increased incorporation of label into 100 000 and 80 000 proteins. After ionophore pulse, the adhering cells retained the ability to differentiate and accumulate myosin. The effect of Ca2+ ionophores upon the rate of protein synthesis is presumably related to increased influx of extracellular Ca2+ with a rise in the Ca2+ concentration of the cytoplasm. We conclude that Ca2+ sensitive mechanisms may regulate the synthesis of a select group of muscle proteins.     

Effect of streptozotocin-diabetes on rat liver asialoglycoprotein receptor turnover: in vivo degradation and in vitro biosynthesis

The metabolic turnover of the Hepatic Binding Protein (HBP) was investigated in streptozotocin-diabetic rats. We have already shown that diabetes induced a decreased ligand binding capacity while the immunoreactive HBP was normal. To explore the eventual modifications due to diabetic state upon the turnover of HBP, we followed the in vivo degradation of HBP and its biosynthesis in vitro. After in vivo labelling with L-[3H] leucine and purification of HBP from rat livers, we found a 20% decrease in diabetic HBP half-life. By in vitro incubations of freshly isolated hepatocytes and a 2 h-pulse in the presence of L-[35S] methionine, we showed that diabetes provokes an increased uptake of L-[35S] methionine in hepatocytes allowing an augmented synthesis of HBP although the L-[35S] methionine incorporation into total proteins was less efficient.     

Alteration of the synthesis of lipoxygenase in the early stages of soybean cotyledon culture

A detailed study of lipoxygenase (EC 1.13.11.12) synthesis in cotyledons of soybean [ Glycine max (L.) Merr. cv. Century] cultured in vitro for up to 40 h showed that synthesis of this protein, measured by in vivo [35S]-methionine labelling in connection with immunological methods and cell-free translation of mRNA, underwent a large transient reduction in the first 4 h of culturing and gradually increased in the following 36 h. Northern blot hybridizations with lipoxygenase cDNA clones showed that the decrease in translational activity was the consequence of a considerable reduction in lipoxygenase mRNA in the cotyledons. From these results we conclude that the transient decline in lipoxygenase synthesis in excised soybean cotyledons is regulated at the RNA level. Similarly judged from the analysis of patterns of uni-dimensional gel electrophoresis, the synthesis of a few other polypeptides decreased during the first 4 h of culture as well, while several others increased; in cotyledons cultured for 20 to 40 h the protein-synthesis pattern had returned to that in freshly excised cotyledons. An acclimation period of ca 1 day seems to be needed for isolated soybean cotyledons to stabilize and to resume regular RNA and protein synthesis.     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

Characterization and hormonal regulation of protein synthesis by the murine epididymis

Protein synthesis and secretion were examined in vitro by incubating minced tissue with [35S]methionine. The incorporation of label into tissue plus medium was linear for the 5 h of incubation. The percentage of available label incorporated into protein increased with the weight of tissue used. Approximately 13% of the label incorporated appeared in the medium after 5 h of incubation. Release of radioactive protein into the medium was characterized by an initial slow release (1-2 h) followed by a more rapid linear release between 3 and 5 h. Polyacrylamide gel electrophoresis revealed that the pattern of radioactive proteins present in the medium was different from and less complex than the tissue proteins. Substantial differences in protein patterns from different epididymal regions could be detected. The caput epididymidis was particularly active in secreting proteins characteristic of this region, whereas the corpus and cauda synthesized and secreted similar proteins. At least one of these proteins characteristic of the caput is stabilized by disulphide bonds. Short-term (9 day) castration resulted in reduced synthesis and secretion of several of these epididymal proteins. Testosterone administered after 9 days of castration reinitiated synthesis of some but not all of these epididymal proteins.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Site of synthesis of the proteins of mammalian mitochondrial ribosomes. Evidence from cultured bovine cells

In order to determine the sites of synthesis of the proteins of the mammalian mitochondrial ribosome (mitoribosome), bovine (MDBK) cells were labeled with [35S]methionine in the presence of inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeling in the absence of cytoplasmic protein synthesis produced a "blank" fluorogram, indicating that there is no mitochondrial product. Additionally, incorporation of [35S]methionine into the enumerated mitoribosomal proteins continued in the absence of mitochondrial protein synthesis. Finally, it was demonstrated that mitoribosomal proteins can be both translated and assembled into complete mitoribosomes in the absence of mitochondrial protein synthesis. These results indicate that in mammals, as opposed to lower eukaryotes, all of the mitoribosomal proteins are products of cytoplasmic protein synthesis.     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.