Transsulphuration and methylation of homocysteine in control and mutant human fibroblasts

The ability of human skin-fibroblasts in monolayer culture to carry out transsulphuration and remethylation of homocysteine has been tested. The conversion of homocyst(e)ine to cyst(e)ine and methionine was studied in control and mutant cells by incubation for 16 h with l-[³⁵S]homocystine. Labelled cysteic acid and methionine sulphone were found in hydrolysates of oxidized cell proteins. The quantities found were dependent on the time of incubation and were used as a measure of cyst(e)ine and methionine formation, respectively. In control cells, labelled cyst(e)ine and labelled methionine were found. In cystathionine β-synthase-deficient cell lines, labelled cyst(e)ine formation was reduced, while labelled methionine formed was similar to that of controls, indicating the role of transsulphuration in the formation of cyst(e)ine observed in control cells. In a 5,10-methylenetetrahydrofolate reductase-deficient cell line, labelled methionine formation was reduced, indicating the role of N-5-methyltetrahydrofolate-requiring methylation of homocysteine in the formation of methionine observed in control cells.     

Severe pathogenic variants of intestinal sucrase-isomaltase interact avidly with the wild type enzyme and negatively impact its function and trafficking

Sucrase-isomaltase (SI) is the major disaccharidase of the small intestine, exhibiting a broad α-glucosidase activity profile. The importance of SI in gut health is typified by the development of sucrose and starch maldigestion in individuals carrying mutations in the SI gene, like in congenital sucrase-isomaltase deficiency (CSID). Common and rare defective SI gene variants (SIGVs) have also been shown to increase the risk of irritable bowel syndrome (IBS) with symptoms and clinical features similar to CSID and also in symptomatic heterozygote carriers. Here, we investigate the impact of the most abundant and highly pathogenic SIGVs that occur in heterozygotes on wild type SI (SI^WT) by adapting an in vitro system that recapitulates SI gene heterozygosity. Our results demonstrate that pathogenic SI mutants interact avidly with SI^WT, negatively impact its enzymatic function, alter the biosynthetic pattern and impair the trafficking behavior of the heterodimer. The in vitro recapitulation of a heterozygous state demonstrates potential for SIGVs to act in a semi-dominant fashion, by further reducing disaccharidase activity via sequestration of the SI^WT copy into an inactive form of the enzymatic heterodimer. This study provides novel insights into the potential role of heterozygosity in the pathophysiology of CSID and IBS.     

Darier disease : A disease model of impaired calcium homeostasis in the skin

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca²⁺ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca²⁺ transport by SERCA2 resulting in decreased ER Ca²⁺ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca²⁺ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Roles of the Mitochondria and Endoplasmic Reticulum in Calcium Elevation during Osmotic Shock in Adrenocortical Cells

Steroid hormones participate in various metabolic processes, and dysfunction of the adrenocortical system leads to numerous pathologies in humans. One of the factors that can influence the secretory properties of adrenocorticocytes is changes in the cell volume observed during osmotic shock. In our study, we tested the hypothesis that osmotic stress modifies intracellular Ca²⁺ signalling and in such a way can influence the secretion of steroids by adrenocorticocytes. The effects of hyperosmotic stress on the cytosolic Ca²⁺ concentration ([Ca] _i ) in cultured adrenocortical cells from the zona fasciculata of the rat adrenals were investigated using the indicator fura-2 technique. Our experiments have shown that exposure of the cells to a hyperosmotic solution caused a decrease in the cell volume, as well as a reversible rise in the [Ca] _i . Calcium-free media partly eliminated [Ca] _i responses. Pretreatment of the cells with thapsigargin or CCCP (blockers of internal calcium stores) significantly decreased the magnitude of responses induced by osmotic stress. These findings indicate that osmotic shock causes an increase in the [Ca] _i in adrenocortical cells, mostly due to depletion of the intracellular stores, and may in such a way stimulate steroidogenesis.     

The change in the electro-rotation of yeast cells effected by silver ions

Pre-treatment of brewer's yeast (Saccharomyces cerevisiae) cells with silver acetate or nitrate at concentrations of 20 nmol/l or higher caused a dramatic increase in the number of cells which rotated in the same direction as the field (‘Co-field rotation’). The change in rotation of single cells correlated very well with the chemically observed loss of potassium induced by Ag⁺. The sensitivity to Ag⁺ was lowered by increasing the cell concentration, and the extent of this change can be used to estimate the binding of Ag⁺ per cell and the limiting sensitivity of the method. The Ag⁺ concentration required to induce a response was found to be increased significantly in the presence of alkali ions (especially K⁺) during the Ag⁺ incubation. The Ag⁺ sensitivity was, therefore, observed to be a function of the type and strength of buffer used in the incubation. Under certain conditions, 1 mM Ca²⁺ increased the Ag⁺ sensitivity. These observations show that the presence or absence of ions that are so common that they are often overlooked may have interesting consequences for the bio-assay of heavy metals.     

Embryogenic and callus-specific proteins in somatic embryogenesis of the grass,Dactylis glomerata L.

Somatic embryogenesis occurs spontaneously in some monocotyledoneous callus and cell suspension cultures maintained in suitable culture conditions. Nevertherless, the processes involved in somatic embryo development, and factors inducing this differentiation, are poorly understood. In order to study the changes in protein composition accompanying embryogenesis in cell suspension cultures of Dactylis glomerata L., embryos of various sizes and “undifferentiated” callus cells were separated and their total cellular protein extracts analyzed by two-dimensional polyacrylamide gel electrophoresis. Several proteins could be identified that are specific for embryos or callus under various culture conditions. Three independent detection methods were employed: silver-staining of proteins, in vivo labeling of proteins with [³⁵S]methionine, and in vitro translation of poly(A)⁺ RNA. All culture conditions tested, including those that induce embryonic proteins in carrot, fail to induce embryonic proteins in D. glomerata callus cells.     

Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes.

Freshly isolated rat hepatocytes contained a high level (30–40 nmol/10⁶ cells) of reduced glutathione (GSH) which decreased steadily upon incubation in an amino acid containing medium lacking cysteine and methionine. This decrease in GSH level was prevented, and turned into a slight increase, when either cysteine, N-acetylcysteine, or methionine was also present in the medium. The amino acid uptake into hepatocytes was more rapid with cysteine than with methionine. Cystine was not taken up, or taken up very slowly, by the cells and could not be used to prevent the decrease in GSH level which occurred in the absence of cysteine and methionine. The level of GSH in hepatocytes freshly isolated from rats pretreated with diethylmaleate was markedly decreased (to ~5 nmol/10⁶ cells) but increased rapidly upon incubation of the cells in a medium containing amino acids including either cysteine, N-acetylcysteine, or methionine. Again, cysteine was taken up into the cells more rapidly than methionine. The rate of uptake of cysteine was moderately enhanced in hepatocytes with a lowered level of intracellular GSH as compared to cells with normal GSH concentration. Exclusion of glutamate and/or glycine from the medium did not markedly affect the rate of resynthesis of GSH by hepatocytes incubated in the presence of exogenously added cysteine or methionine. Incubation of hepatocytes with bromobenzene in an amino acid-containing medium lacking cysteine and methionine resulted in accelerated cell damage. Addition of either cysteine, N-acetylcysteine, or methionine to the medium caused a decrease in bromobenzene toxicity. The protective effect was dependent, however, on the time of addition of the amino acid to the incubate; e.g., the effect on bromobenzene toxicity was greatly reduced when either cysteine or methionine was added after 1 h of preincubation of the hepatocytes with bromobenzene as compared to addition at zero time. This decrease in protective effect in bromobenzene-exposed cells was related to a similar decrease in the rate of uptake of cysteine and methionine into hepatocytes preincubated with bromobenzene. The rate of uptake, and incorporation into cellular protein, of leucine was also markedly inhibited in hepatocytes preincubated with bromobenzene. In contrast, there was no measurable change in the rate of release of leucine from cellular protein as a result of incubation of hepatocytes with bromobenzene. It is concluded that the presence of cysteine, N-acetylcysteine, or methionine in the medium protects hepatocytes from bromobenzene toxicity by providing intracellular cysteine for GSH biosynthesis and suggested that an inhibitory effect on amino acid uptake may contribute to the cytotoxicity of bromobenzene in hepatocytes.     

Anion channels in giant liposomes made of endoplasmic reticulum vesicles from rat exocrine pancreas

Summary Using the method of dehydration and rehydration, rough endoplasmic reticulum (RER) vesicles, isolated by differential centrifugation, can be enlarged to giant liposomes with diameters ranging from 5 to 200 m. Patch-clamp studies on these giant RER liposomes revealed the existence of a channel with a mean conductance of 260±7 pS (n=23; 140 mmol/liter KCl on both sides). The channel is about four times more permeable for Cl^– than for K⁺. Its activity is strongly voltage regulated. At low potentials (±20 mV) the channel is predominantly in its open state with an open probability near 1.0, whereas it closes permanently at high positive and negative voltages (±70 mV). The channel activity is not influenced by changing the free Ca²⁺ concentration from 1 mmol/liter to less than 10^–9 mol/liter on either side, and is also not affected by typical Cl^–-channel blockers like diphenylamine-2-carboxylate (DPC, 1 mmol/liter) or 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mmol/liter). Another chloride channel with a singlechannel conductance of 79±6 pS (n=4) was less frequently observed. In the potential range of –80 to +40 mV this channel displayed no voltage-dependent gating. We assume that these anion channels are involved in the maintenance of electroneutrality during Ca²⁺ uptake in the RER.     

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2^+/− mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.     

Regulation of SERCA pumps expression in diabetes

Cytosolic calcium concentration ([Ca²⁺]_c) is fundamental for regulation of many cellular processes such metabolism, proliferation, muscle contraction, cell signaling and insulin secretion. In resting conditions, the sarco/endoplasmic reticulum (ER/SR) Ca²⁺ ATPase's (SERCA) transport Ca²⁺ from the cytosol to the ER or SR lumen, maintaining the resting [Ca²⁺]_c about 25–100 nM. A reduced activity and expression of SERCA2 protein have been described in heart failure and diabetic cardiomyopathy, resulting in an altered Ca²⁺ handling and cardiac contractility. In the diabetic pancreas, there has been reported reduction in SERCA2b and SERCA3 expression in β-cells, resulting in diminished insulin secretion. Evidence obtained from different diabetes models has suggested a role for advanced glycation end products formation, oxidative stress and increased O-GlcNAcylation in the lowered SERCA2 expression observed in diabetic cardiomyopathy. However, the role of SERCA2 down-regulation in the pathophysiology of diabetes mellitus and diabetic cardiomyopathy is not yet well described. In this review, we make a comprehensive analysis of the current knowledge of the role of the SERCA pumps in the pathophysiology of insulin-dependent diabetes mellitus type 1 (TIDM) and type 2 (T2DM) in the heart and β-cells in the pancreas.     

Characterization of photodynamic therapy responses elicited in A431 cells containing intracellular organelle‐localized photofrin

Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363–375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle‐localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2–8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA‐sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin‐PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca²⁺‐ATPase inhibitor that perturbs Ca²⁺ homeostasis, suggesting a role for Ca²⁺ in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT. J. Cell. Biochem. 111: 821–833, 2010. © 2010 Wiley‐Liss, Inc.     

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis

Mycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca²⁺ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.     

Invertase and auxin-induced elongation in internodal segments of Phaseolus vulgaris

A close positive correlation was observed between segment elongation and the specific activity of soluble acid invertase in stem segments of P. vulgaris incubated for 21 hr in the presence of IAA or of several synthetic auxins and auxin analogues. Optimum concentrations for the stimulation of growth and invertase activity were similar and varied from 10^?6 M (2,4-D) through 10^?5 M (IAA, IBA, α-NAA, β-NAA) to greater than 10^?4 (IPA, PoAA, trans-cinnamic acid). The weak activity of trans-cinnamic acid, a competitive inhibitor of auxin action, may have resulted from cis-trans isomerization during incubation. The concentration of hexose sugars in the segments fell during incubation in the presence of auxin, the greatest decline in hexose concentration occurring in the presence of compounds exhibiting the greatest stimulation of growth.     

Mutation of mitochondrial ATP8 gene improves hepatic energy status in a murine model of acute endotoxemic liver failure

AimsMitochondria not only generate and modulate bioenergy but also serve as biosensors for oxidative stress, and eventually become effector organelles for cell viability. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene affects the course of endotoxemic acute liver failure.Main methodsC57BL/6J (ATP8 wild type) and C57BL/6J-mt^FVB/N (ATP8 mutant) mice were challenged with d-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) for induction of acute liver failure, and studied 6 h thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and levels of hepatocellular apoptosis, hepatic adenosine nucleotides and oxidative stress. Additionally, survival rates were assessed.Key findingsInduction of endotoxemic liver failure provoked marked liver damage, which was coexistent with a drop of total adenosine nucleotide levels and increased oxidative stress. Of interest, oxidative stress was higher in the GalN/LPS challenged ATP8 mutants compared to wild types. Concomitantly, adenosine triphosphate (ATP) levels in livers of mice carrying the ATP8 mutation remained higher than those in wild type mice. As net result, ATP8 mutants showed lower transaminase release and a tendency to better survival rate upon GalN/LPS exposure compared to wild types.SignificanceOur findings demonstrate that mutation in the ATPase subunit-8 partially protects mice against endotoxemic stress, most probably due to better hepatic energy status despite elevated oxidative stress. Thus, modulating mitochondrial function to preserve bioenergetic status may be an effective strategy to protect against sepsis-induced multiorgan dysfunction.     

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen‐activated protein kinases in pancreatic tumour AR42J cells

Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3(2H)‐one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti‐inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free‐Ca²⁺ concentration ([Ca²⁺]_c), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen‐activated protein kinases were analyzed. Our results show that ebselen evoked a concentration‐dependent increase in [Ca²⁺]_c. The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin‐evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X‐box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen‐activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells.     

Effect of theophylline and Na+ on methionine influx in Na+-depleted intestine

The unidirectional influx of methionine into the brush border epithelium of chicken jejunum has been studied. Tissues leached of Na⁺ transport methionine from a medium devoid of Na⁺ with reduced apparent affinity (K_t) and maximal flux (J_max). Addition of Na⁺ to the medium during a 1-min incubation with substrate, or during a 30-min preincubation, restored K_t but affected J_max slightly. Theophylline was found to maintain J_max in the absence of Na⁺. Essentially complete restoration of K_t and J_max could be attained when theophylline-treated tissue was exposed to Na⁺ for 30 min. Influx from a Na⁺ medium was unaffected by theophylline pretreatment in Na⁺-containing buffer. K_t was increased without an effet upon J_max when influx was studied from choline medium following preincubation in Na⁺.Modifiers of tissue cyclic AMP levels were investigated in conjunction with theophylline. Histamine and carbachol were found to inhibit theophylline-stimulated transport. Secretin was found to stimulate influx in Na⁺-leached tissue, but did not potentiate the theophylline effect. Amino acids in the incubation medium inhibited theophylline-stimulated influx, whereas preloaded lysine or methionine had no effect.The results are interpreted in terms of a model which envisions roles for cellular and external Na⁺ and for cyclic AMP in the activation and regulation of amino acid transport in intestine.     

Altered distribution profiles of endoplasmic reticulum subfractions after incubation of Krebs II ascites cells with different concentrations of cytochalasin B

Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5–10 μg ml^?1) resulted in an increased yield of three ER membrane subfractions — heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by ³H-choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using ¹³C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (K_m approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h⁻¹ g⁻¹ cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. ³¹P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h⁻¹ g⁻¹ cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h⁻¹ g⁻¹ cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.