Involvement of cyclic GMP in the initial stage of hepatocytes proliferation.

A transient rise in cyclic guanosine 3' : 5' monophosphate (c-GMP) in the liver was observed in rats in vivo 10--20 min after partial hepatectomy. A similar increase in c-GMP in the liver was also found in rats in vivo 15 min after infusion of TGH solution (a mixture of triiodothyronine, glucagon, and heparin). In both cases, inductions of ornithine decarboxylase [EC 4.1.1.17] and tyrosine aminotransferase [EC 2.6.1.5] were found 4 hr after the beginning of the experiments. Later, 22 hr after the surgical intervention or hormone infusion, thymidine kinase [EC 2.7.1.21] was activated and liver slices were able to incorporate [3H]thymidine into DNA. These biochemical phenomena were observed commonly in regenerating liver as well as in the liver of rats infused with TGH solution. c-GMP, but not c-AMP, could induce ornithine decarboxylase and tyrosine aminotransferase in isolated, perfused liver.     

Possible cyclic AMP-dependence of the prereplicative surge of cytosolic calmodulin in proliferatively activated rat liver cells

The infusion of a solution containing triiodothyronine, amino acids, glucagon, and heparin (TAGH solution) triggered rat liver cell proliferation. It also induced a transient prereplicative surge of cytosolic calmodulin (between 6 and 20 hr postinfusion) similar to that observed in liver cells proliferatively activated by partial hepatectomy. The injection of the beta-adrenergic blocker dl-propanolol (20 mg/kg of body weight) at the time of the infusion prevented this transient rise of cytosolic calmodulin and also inhibited the early prereplicative surge of total liver cyclic AMP, which usually occurred between 1 and 4 hr after infusion. Propanolol also inhibited the early prereplicative surge of cyclic AMP and the increase of calmodulin in liver cells proliferatively activated by partial hepatectomy. The infusion of a solution containing cyclic AMP (5 mumoles) and theophylline (10 mg) into normal rats produced an increase of cytosolic calmodulin similar to that observed after infusion of TAGH solution or after partial hepatectomy. Thus it seems that the prereplicative rise of cytosolic calmodulin observed in proliferatively activated liver cells may be regulated by the early prereplicative surge of cyclic AMP.     

Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.

A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.     

Effect of cobalt on heme biosynthesis in rat liver and spleen.

1. Succinate-cytochrome c reductase activity in rat liver decreased to about 60% of the control value after a single injection of cobalt or in a steady state of intoxication, but the activity in the spleen was unaltered. 2. Incorporation of radioactive glycine and 5-aminolevulinate into heme of the liver was markedly inhibited by cobalt treatment. 3. 5-Aminolevulinate synthase [EC 2.3.1.37] activity in the liver decreased to 40% of the control value 4 hr after cobalt injection, and completely recovered 20 hr later. Phenylhydrazine-induced 5-aminolevulinate synthase activity in the spleen was not decreased by cobalt injection. 4. Porphobilinogen synthase [EC 4.2.1.24] activity in the liver decreased and reached its minimum value (42% of the control) 12 hr after cobalt injection. On the other hand, the activity in the spleen showed a marked increase 24 hr after coblat injection. 5. Ferrochelatase [EC 4.99.1.1] activity in the liver was essentially unaltered by cobalt treatment, while the activity in the spleen was elevated dramatically after 24 hr. 6. Concentrations of cobalt after a single injection were about 0.3 mM and 0.03 mM in the liver and spleen, respectively. 7. Inhibitions of 5-aminolevulinate synthase and porphobilinogen synthase activities by cobalt in vitro were not as marked as expected from in vivo experiments.     

Metabolism of sialic acid in regenerating rat liver.

In regenerating rat liver slices 24 h after partial hepatectomy, the incorporation of [1-14C]glucosamine into 'free sialic acid' (N-acetylneuraminic acid + CMP-N-acetylneuraminic acid) decreased to below 50% of the control values and the incorporation into protein-bound sialic acid decreased to the same extent. The incorporation of [14C]glucosamine into 'free sialic acid' decreased during the period from 6 to 47 h after hepatectomy, showing a minimum at 12 h, and recovered to the control value by 96 h. At 12 h, the activities of UDP-N-acetylglucosamine 2-epimerase (UDP-2-acetoamido-2-deoxy-D-glucose 2-epimerase, EC 5.1.3.14) and N-acyl-D-mannosamine kinase (ATP: 2-acylamino-2-deoxy-D-mannose 6-phosphotransferase, EC 2.7.1.60) in the liver were significantly decreased. The amount of protein-bound sialic acid in the liver was not changed after partial hepatectomy, but the amount in plasma was changed, with a similar pattern to that of the incorporation of [14C]glucosamine into slice 'free sialic acid'. These results indicate that the synthesis of sialic acid in the liver much decreases in the early stage of regeneration and that this may be correlated with the decreased synthesis of plasma sialoglycoproteins.     

Blockage of prereplicative progression by cytochalasin D in serum-stimulated GC-7 cells

When growth-arrested GC-7 cells, a cell line from African green monkey kidney, are stimulated with 10% calf serum, they enter S phase 14-15 h later. Cytochalasin D at 0.6 micrograms/ml blocks the entrance into S phase, and inhibits, though only partially, the increase in protein synthesis after serum stimulation. Since partial inhibition of protein synthesis by cycloheximide interferes with accumulation of labile proteins and thus blocks the entrance of serum-stimulated cells into S phase, the effects of these two inhibitors are compared. Cytochalasin D at lower concentrations reduced the rate of entry into S phase without affecting the length of the prereplicative phase, whereas cycloheximide extended the prereplicative phase dose dependently without affecting the rate of entry into S phase. Cytochalasin D affected neither individual [35S]methionine-labeled spots on two-dimensional polyacrylamide-gel nor degradation of cellular proteins. These results indicate that cytochalasin D, though it interferes with protein synthesis, blocks prereplicative progression of serum-stimulated GC-7 cells in a different manner than cycloheximide.     

Effect of hydrazine exposure on hepatic triacylglycerol biosynthesis.

Acute hydrazine exposure elevated rat liver triacylglycerol content and produced a rapid rise in triacylglycerol production from sn-[1,3-14C]glycerol 3-phosphate by liver homogenate and microsomal fractions. Hydrazine treatment also increased the incorporation of [1,3-14C]glycerol into hepatic triacylglycerol by the intact animal. Homogenates of hepatocyte monolayers exposed to hydrazine in vitro also exhibited an increased capacity to form triacylglycerol from sn-[1,3-14C]glycerol 3-phosphate. Hydrazine-dependent increases in hepatic triacylglycerol production measured in vitro correlated well with an increase in microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) activity. Therefore, the fatty liver associated with hydrazine exposure may be explained in part by a rise in the enzymatic capacity of hepatic triacylglycerol biosynthesis.     

Biphasic increase in ornithine decarboxylase and its relationship with thymidine kinase and DNA synthesis in liver: Effects of dietary protein and amino acid mixture

In rats, feeding protein free diet for 4 days followed by starvation and then high protein diet induced a biphasic ornithine decarboxylase (EC 4.1.1.17) activity, prolonged thymidine kinase (EC 2.7.1.21) activity and DNA synthesis. In contrast feeding a diet containing casein-equivalent amino acid mixture induced a monophasic ornithine decarboxylase activity, short-lived thymidine kinase activity and DNA synthesis. To maintain prolonged thymidine kinase activity and DNA synthesis high protein diet must be given in the early part of the prereplicative period.     

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration

Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.     

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.     

Peroxide-Scavenging Systems during Cold Acclimation of Apple Callus in Culture

The relationship between peroxide-scavenging systems and coldacclimation was studied in apple callus in culture during acclimationunder artificial conditions. Unacclimated callus did not survivefreezing at –10?C, whereas callus acclimated at 0?C exhibitedgradually increased resistance to freezing and, after acclimationfor 20 days, it survived at temperatures as low as –15–C.During acclimation of callus, there was an immediate and abruptincrease in the activities of ascorbate peroxidase (EC 1.11.1.11 [EC] ),peroxidase (EC 1.11.1.7 [EC] ) and catalase (EC 1.11.1.6 [EC] ), which reachedmaximum values after acclimation for 10 days, at the same timeas the very beginning of the increase in cold hardiness wasobserved. An increase in the activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) occurred during the first 5 daysof cold treatment. The activities of glucose-6-phosphate dehydrogenase(EC 1.1.1.49 [EC] ), hexokinase (EC 2.7.1.1 [EC] ), glutathione reductase(EC 1.6.4.2 [EC] ), glutathione peroxidase (EC 1.11.1.9 [EC] ) and dehydro-ascorbatereductase (EC 1.8.5.1 [EC] ) increased gradually during the cold treatment.In contrast, the activity of glucosephosphate isomerase (EC5.3.1.9 [EC] ) decreased gradually during acclimation. Furthermore,during acclimation, the levels of glucose-6-phosphate, fructose-6-phosphateand glucose-1-phosphate increased slowly and steadily, and thelevels of GSH and ascorbate remained at consistently higherlevels. In addition, acclimation caused marked cytological changes.The most striking of these changes was the microvacuolationand thickening of the cell wall. These results indicate thatthe enhancement of peroxide-scavenging systems at the time ofcold acclimation proceeds in two stages: during the first stage,the enzymatic activities involved in the degradation of peroxides(i.e., the activities of ascorbate peroxidase, peroxidase andcatalase) increase; and, in the second stage, an alternativeenzymatic system develops for detoxification of peroxides, coupledwith the pentose phosphate cycle. (Received July 20, 1990; Accepted April 16, 1991)     

Studies on a rat-liver cell-sap protein yielding 3-[32P]-phosphohistidine after incubation with [32P]ATP and alkaline hydrolysis. Identification of the protein as ATP citrate lyase

1. The rat-liver cell-sap material from which 3-[32P]phosphohistidine was previously isolated after incubation with [gamma-32P]ATP and alkaline hydrolysis, was shown to increase about 6-fold on a high-carbohydrate diet. This increase in 32P labelling corresponded to the increase in ATP citrate lyase activity of livers of rats fed on a high-carbohydrate diet, as reported by others. 2. ATP citrate lyase [ATP:citrate oxaloacetate-lyase (CoA-acetylating and ATP-dephopshorylating), EC 4.1.3.8] was purified from rat liver essentially according to the method of Plowman and Cleland (J. Biol. Chem., 242 (1967) 4239). The purified enzyme was incubated for a short time at 0 degree with [gamma-32P]ATP in the presence of 20 mM magnesium acetate. The phosphorylated protein was hydrolysed in alkali and the main part of the radioactivity was identified as 3-[32P]phosphohistidine. The identity of the phosphorylated amino acid was established by Dowex-1 chromatography, paper electrophoresis, paper chromatography and by analysis of the stability to acid. 3. It is concluded from these and previous results from this laboratory that ATP citrate lyase and nucleoside diphosphate kinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) account for most of the normal rat-liver cell-sap protein which is rapidly phosphorylated by ATP.     

Studies on the turnover rates of ornithine aminotransferase in Morris hepatoma 44 and host liver.

The ornithine aminotransferase [EC 2.6.1.13] content of Morris hepatoma 44 is about 15 times higher than that in normal liver. The turnover rates of ornithine amino-transferase in hepatoma 44 and host liver were determined using L-[14C]leucine. Studies on the incorporation of radioactive leucine into ornithine aminotransferase in rats bearing hepatoma 44 showed that the rate of synthesis of this enzyme in the hepatoma was about 5-fold higher than that in host liver. The half-life of ornithine aminotransferase in host liver was 0.98 day, which was the same as that in normal liver, whereas that in hepatoma 44 was 3.5 days. The rate constant of degradation of ornithine aminotransferase in hepatoma 44 was significantly less than that in host liver. These results show that the high ornithine aminotransferase content of hepatoma 44 is due to both increase in its rate of synthesis and decrease in its rate of degradation.     

Metabolic pattern of polymorphonuclear leucocytes induced by trypsin-digested microsomes.

The addition of trypsin [EC 3.4.21.4]-digested liver microsimes induced cyanideinsensitive respiration in guinea pig polymorphonuclear leucocytes with concomitant acceleration of the hexose monophosphate oxidative pathway. The respiration was insensitive to inhibitors of mitochondrial respiration but sensitive to glycolytic inhibitors. These metabolic alterations are similar to those associated with phagocytosis, though the digested mocrosomes were apparently not taken up by the cells and prpbably trigger the netabolic changes by interaction with the cellular membrane. Intact microsomes or microsomes treated with chymotrypsin [EC 3.4.21.1], bacterial proteinase, ribonuclease [EC 3.1.4.22], or neuraminidase [EC 3.2.1.18] could not induce such respiration.     

Aspects of carnitine ester metabolism in sheep liver

1. Carnitine acetyltransferase (EC 2.3.1.7) activity in sheep liver mitochondria was 76nmol/min per mg of protein, in contrast with 1.7 for rat liver mitochondria. The activity in bovine liver mitochondria was comparable with that of sheep liver mitochondria. Carnitine palmitoyltransferase activity was the same in both sheep and rat liver mitochondria. 2. The [free carnitine]/[acetylcarnitine] ratio in sheep liver ranged from 6:1 for animals fed ad libitum on lucerne to approx. 1:1 for animals grazed on open pastures. This change in ratio appeared to reflect the ratio of propionic acid to acetic acid produced in the rumen of the sheep under the two dietary conditions. 3. In sheep starved for 7 days the [free carnitine]/[acetylcarnitine] ratio in the liver was 0.46:1. The increase in acetylcarnitine on starvation was not at the expense of free carnitine, as the amounts of free carnitine and total acid-soluble carnitine rose approximately fivefold on starvation. An even more dramatic increase in total acid-soluble carnitine of the liver was seen in an alloxan-diabetic sheep. 4. The [free CoA]/[acetyl-CoA] ratio in the liver ranged from 1:1 in the sheep fed on lucerne to 0.34:1 for animals starved for 7 days. 5. The importance of carnitine acetyltransferase in sheep liver and its role in relieving ;acetyl pressure' on the CoA system is discussed.     

Synthesis of Phytochelatins and Homo-Phytochelatins in Pisum sativum L

In the roots of pea plants (Pisum sativum L.) cultivated with 20 [mu]M CdCl2 for 3 d, synthesis of phytochelatins [PCs or ([gamma]EC)nG, where [gamma]EC is [gamma]glutamylcysteine and G is glycine] and homophytochelatins [h-PCs, ([gamma]EC)n[beta]-alanine] is accompanied by a drastic decrease in glutathione (GSH) content, but an increase in homoglutathione (h-GSH) content. In contrast, the in vitro activity of GSH synthetase increases 5-fold, whereas h-GSH synthetase activity increases regardless of Cd exposure. The consititutive enzyme PC synthase, which catalyzes the transfer of the [gamma]-EC moiety of GSH to an acceptor GSH molecule thus producing ([gamma]EC)2G, is activated by heavy metals, with Cd and Cu being strong activators and Zn being a very poor activator. Using h-GSH or hm-GSH for substrate, the synthesis rate of([gamma]EC)2[beta]-alanine and [gamma]EC)2-serine is only 2.4 and 0.3%, respectively, of the sythesis rate of ([gamma]EC)2G with GSH as substrate. However, in the presence of a constant GSH level, increasing the concentration of h-GSH or hm-GSH results in increased synthesis of ([gamma]EC)2[beta]-alanine or ([gamma]EC)2-serine, respecively; simultaneously, the synthesis of ([gamma]EC)2G is inhibited. [gamma]EC is not a substrate of PC synthase. These results are best explained by assuming that PC synthase has a [gamma]EC donor binding site, which is very specific for GSH, and a [gamma]EC acceptor binding site, which is less specific and accepts several tripeptides, namely GSH, h-GSH, and hm-GSH.     

Purification and partial characterization of phosphatidylinositol-4-phosphate kinase from rat liver plasma membranes. Further evidence for a stimulatory G-protein

Phosphatidylinositol 4-phosphate (PIP) kinase (E.C. 2.7.1.68) has been purified about 1200-fold from rat liver plasma membranes, taking advantage of affinity chromatography on quercetin-Sepharose as a novel step. The purified PIP kinase showed no contamination by the following enzyme activities: phosphatidylinositol (PI) kinase (EC 2.7.1.67), protein kinase C (EC 2.7.1.-), diacylglycerol kinase (EC 2.7.1.-), phospholipase C (EC 3.1.4.11), protein-tyrosine kinase (EC 2.7.1.112), alkaline phosphatase (EC 3.1.3.1), triphosphoinositide phosphomonoesterase (EC 3.1.3.36), adenylate kinase (EC 2.7.4.3) and cAMP-dependent protein kinase (EC 2.7.1.37). The liver membrane enzyme requires high Mg2+ concentrations with a KM value of 10 mM. Ca2+ or Mn2+ could replace Mg2+ to a certain, though small, extent. Apparent KM values with respect to PIP and ATP were 10 and 65 microM, respectively. GTP was slightly utilized by the kinase as phosphate donor while CTP was not. Quercetin inhibited the enzyme with Ki = 34 microM. Extending our previous observations (Urumow, T. and Wieland, O.H. (1986) FEBS Lett. 207, 253-257 and Urumow, T. and Wieland, O.H. (1988) Biochim. Biophys. Acta 972, 232-238) [gamma S]pppG still stimulated the PIP kinase in extracts of solubilized liver membranes. 20-40% (NH4)2SO4 precipitation of the membrane extracts yielded a fraction that contained the bulk of enzyme activity but did not respond to stimulation by [gamma S]pppG any longer. This was restored by recombination with a protein fraction collected at 40-70% (NH4)2SO4 saturation, presumably containing a GTP binding protein and/or some other factor separated from the PIP kinase. In the reconstituted system [gamma S]pppG stimulated PIP kinase in a concentration dependent manner with maximal activation at 5 microM. This effect was not mimicked by [gamma S]pppA and was blocked by [beta S]ppG. These results strongly support our view that in liver membranes PIP kinase is regulated by a G-protein.     

Plasma membrane lipid metabolism of petunia petals during senescence

The specific activities of 6 enzymes, which are involved in the synthesis and catabolism of membrane lipids, were monitored in plasma membranes isolated from petunia petals during senescence. These included phosphatidylinositol (PI) kinase (EC 2.7.1.67), phosphatidylinositol monophosphate (PIP) kinase (EC 2.7.1.68). diacylglycerol (DAG) kinase (EC 2.7.1.107), phospholipase A (EC 3.1.1.4) and PIP- and PIP₂-phospholipase C˙(EC 3.1.4.3). Using endogenous substrate, the [³²P]PA and [³²P]PIP₂ formation increased to 140 and 200%, respectively, of the day 1 value by 4 days after harvest. There was no significant change in [³²P]PIP formation during the same time period. On the fifth day the petals wilted and the [³²P]PA and [³²P]PIP formation declined significantly. In contrast, the [³²P]PIP₂ formation remained high in the day 5 petals. When the lipid kinase activities were assayed in the membranes in the presence of exogenous substrate the specific activity of all of the enzymes increased. and the changes in [³²P]PA production over the 5-day period were similar to those observed with endogenous substrate. When exogenous PI and PIP were added, however, there was no longer an increase in [³²P]PIP₂ formation by plasma membranes of day 4 petals and [³²P]PIP formation significantly decreased. The relative decrease in PIP and PIP₂ formation by day 4 membranes when exogenous substrate was added may have resulted from differences in the lipase activities in the day 1 and day 4 membranes. The plasma membrane A-type phospholipase activity increased throughout the 5 day period, and phospholipase C activity increased two-fold between day 1 and day 4. Such changes in the metabolism of the plasma membrane lipids during flower senescence would affect the ability of the petals to use inositol phospholipid-based signal transduction pathways.     

The Development of Activities of Some Enzymes Concerned with Glycollate Metabolism in Greening Bean Leaves

The activities of phosphoglycollate phosphatase (EC 3.1.3.18 [EC] ),glycollate oxidase (EC 1.1.3.1 [EC] .). catalase (EC 1.11.1.6 [EC] ), theperoxisomal NADH-glyoxylate reductase (EC 1.1.1.26 [EC] ) which isconsidered to function as a hydroxypyruvate reductase in theperoxisomes, and the chloro-plastic NADPH-dependent glyoxylatereductaae, have been measured in extracts prepared from 14-d-olddark-grown bean leaves during the course of their greening inresponse to exposure to continuous illumination. All of theenzymes were found in the dark-grown leaves and on a per-leafbasis the activities increased from 6- to 12-fold with the exceptionof a 2–3-fold increase of NADPH-dependent glyoxylate reductaseduring 96-h greening, while the activities either remained constantor declined during similar periods in darkness. Initial lagperiods were evident before the illumination-induced increasesin enzyme activities. As D-threo-chloramphenicol did not affectthe increase in activity of any of these enzymes it would appearthat the increases were in no way dependent on protein synthesisby 70S ribosomes, or on the development of photosynthetic activity.