Post-mortem visualization of peroxisomes in rat and in human liver

Summary This paper describes spontaneous post-mortem changes of peroxisomal staining in normal liver and kidney of rats and in human autopsy liver. At room temperature, regional staining loss is observed at 18h after death in rat kidney, at 24h in human liver and at 48 h in rat liver. Preservation at 4°C delays this phenomenon. In human liver, the peroxisomal volume density is decreased at both temperatures at 48 h. After freezing of fresh tissue in dry ice, peroxisomal staining is decreased homogeneously. Under the electron microscope, peroxisomal alterations suggest a loss of catalase activity. These changes do not necessarily preclude the study of peroxisomal features since, even after 48 h at room temperature, peroxisomes are still well stained in the less affected regions. Catalase and three -oxidation enzymes, namely acyl-CoA oxidase, bifunctional protein (with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) and 3-oxoacyl-CoA thiolase, could be visualized immunocytochemically in human autopsy livers up to 48 h after death. However, the study of certain peroxisomal features such as catalase activity and peroxisomal distribution, may be hampered as the post-mortem period is prolonged.     

Modification of Proteins in Endothelial Cell Death during Oxidative Stress

Exposure of bovine aortic endothelial cells in vitro to oxidative stress causes a cascade of changes in cell function, culminating in cell death if the stress is sufficiently severe. Oxidative modification of proteins, as measured by the reaction of 2,4-dinitrophenylhydrazine with carbonyl groups of oxidized proteins, increased three- to fourfold in endothelial cells exposed to hydrogen peroxide or to a xanthine/xanthine oxidase system. The increase in oxidative modification of protein occurred rapidly, preceding loss of cellular ATP and eventual cell death. Oxidative modification of protein was paralleled by loss of activity of the key metabolic enzymes, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The finding that oxidative modification of protein is an early event following oxidative stress suggests that oxidative modification of protein is not only a marker for oxidative damage but also a causal factor in oxidative injury. Published by Elsevier Science Inc.     

Stability of neuronal and glial marker enzymes in post-mortem rat brain

The enzymatic activities in post-mortem rat brain kept at 4°C and at 25°C were determined for a number of enzymes localized in specific cell types in the central nervous system. Choline acetyltransferase (CAT), glycerol-3-phosphate dehydrogenase (GPDH), glutamine synthetase (GS), lactate dehydrogenase (LDH) and 2,3-cyclic nucleotide phosphohydrolase (CNPase) were found to be very stable at both 4°C and 25°C with only slight, if any, losses of activity being seen even at periods as long as 72 hr. Glutamic acid decarboxylase (GAD) activity was less stable than that of the other enzymes. In brains kept at 4°C GAD activity was stable out to 24 hr after which it began to decline rapidly to 65% of control at 72 hr. In brains kept at 25°C, GAD activity was stable for 6–8 hr and then began to steadily decline to 58% of control at 24 hr and 29% of control at 72 hr. Assuming that these enzymes have similar stabilities in post-mortem human brain, the effect of post-mortem delay in processing tissues may be of lesser significance than other factors with regard to the measured enzyme activities in human brain samples.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

The post-mortem stability of certain oxidative enzymes in brain and spinal cord

Summary An investigation has been carried out on the stability of several enzymes in portions of rabbit brain and spinal cord kept at controlled temperatures between 22 and 37° C for periods up to 24 hours before processing for enzyme activity. The enzymes studied were NAD diaphorase, succinate, lactate, glutamate and glucose-6-phosphate dehydrogenases, and monoamine oxidase. One-wavelength plug cytophotometric measurements of enzyme activity were carried out on Purkinje cells, neuropil of the granular layer of the cerebellar cortex and on anterior horn cells.Succinate dehydrogenase activity proved to be stable after 24 hours post-mortem exposure at 37°C. Lactate dehydrogenase, NAD diaphorase and monoamine oxidase activities were less stable at the higher temperatures but were stable at 22°C. Glutamate and glucose-6-phosphate dehydrogenase activities fell significantly with exposure at 22°C. It thus appears possible to make valid histochemical measurements of the activities of certain oxidative enzymes in selected post-mortem brain material.This research was aided by a grant from the National Health and Medical Research Council of Australia.     

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration

Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.     

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.     

Alteration in tRNA Methyltransferase Activity in Mengovirus Infection: Host Range Specificity

The tRNA methyltransferase activity in mengovirus-infected L cells, HeLa cells, and Maden Derby bovine kidney cells has been examined during the course of infection. The first two cell lines yield a productive infection, but have different kinetics of inhibition of host RNA synthesis, whereas the bovine kidney cells are a restrictive host. In infected L cells the enzymes show altered capacity and base specificity throughout the infection. In infected HeLa cells and in infected bovine kidney cells less marked changes were seen. No inhibitors or activators of the enzymes were detected in any of the infected cell lines. Labeling experiments in infected cells indicated that in infected L cells synthesis of RNA was inhibited to a greater degree than was methylation of RNA. The consequence of this would be a hypermethylation of RNA. The methylated derivatives synthesized in infected L cells showed changes in relative proportions. Infection of HeLa cells and bovine kidney cells did not show such marked effects on methylation of RNA.     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.     

Overexpression of Pyruvate Dehydrogenase Kinase 1 and Lactate Dehydrogenase A in Nerve Cells Confers Resistance to Amyloid β and Other Toxins by Decreasing Mitochondrial Respiration and Reactive Oxygen Species Production

We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease.     

GAPDH binds to active Akt,leading to Bcl-xL increase and escape from caspase-independent cell death

Increased glucose catabolism and resistance to cell death are hallmarks of cancers, but the link between them remains elusive. Remarkably, under conditions where caspases are inhibited, the process of cell death is delayed but rarely blocked, leading to the occurrence of caspase-independent cell death (CICD). Escape from CICD is particularly relevant in the context of cancer as apoptosis inhibition only is often not sufficient to allow oncogenic transformation. While most glycolytic enzymes are overexpressed in tumors, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of particular interest as it can allow cells to recover from CICD. Here, we show that GAPDH, but no other glycolytic enzymes tested, when overexpressed could bind to active Akt and limit its dephosphorylation. Active Akt prevents FoxO nuclear localization, which precludes Bcl-6 expression and leads to Bcl-xL overexpression. The GAPDH-dependent Bcl-xL overexpression is able to protect a subset of mitochondria from permeabilization that are required for cellular survival from CICD. Thus, our work suggests that GAPDH overexpression could induce Bcl-xL overexpression and protect cells from CICD-induced chemotherapy through preservation of intact mitochondria that may facilitate tumor survival and chemotherapeutic resistance.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Physiological community ecology: variation in metabolic activity of ecologically important rocky intertidal invertebrates along environmental gradients

Rocky intertidal invertebrates live in heterogeneous habitatscharacterized by steep gradients in wave activity, tidal flux,temperature, food quality and food availability. These environmentalfactors impact metabolic activity via changes in energy inputand stress-induced alteration of energetic demands. For keystonespecies, small environmentally induced shifts in metabolic activitymay lead to disproportionately large impacts on community structurevia changes in growth or survival of these key species. Herewe use biochemical indicators to assess how natural differencesin wave exposure, temperature and food availability may affectmetabolic activity of mussels, barnacles, whelks and sea starsliving at rocky intertidal sites with different physical andoceanographic characteristics. We show that oxygen consumptionrate is correlated with the activity of key metabolic enzymes(e.g., citrate synthase and malate dehydrogenase) for some intertidalspecies, and concentrations of these enzymes in certain tissuesare lower for starved individuals than for those that are wellfed. We also show that the ratio of RNA to DNA (an index ofprotein synthetic capacity) is highly variable in nature andcorrelates with short-term changes in food availability. Wealso observed striking patterns in enzyme activity and RNA/DNAin nature, which are related to differences in rocky intertidalcommunity structure. Differences among species and habitatsare most pronounced in summer and are linked to high nearshoreproductivity at sites favored by suspension feeders and to exposureto stressful low-tide air temperatures in areas of low wavesplash. These studies illustrate the great promise of usingbiochemical indicators to test ecological models, which predictchanges in community structure along environmental gradients.Our results also suggest that biochemical indices must be carefullyvalidated with laboratory studies, so that the indicator selectedis likely to respond to the environmental variables of interest.     

Biochemical and enzymic changes during erythrocyte differentiation. The significance of the final cell division.

1. The haemoglobin content of developing erythroblasts was shown to increase rapidly when the cells completed the final cell division of erythroid development and passed from the dividing into the non-dividing cell compartment. 2. The activity of carbonic anhydrase was measured and shown to increase continually throughout erythroid differentiation. The activity increased most rapidly in the polychromatic stage. 3. Catalase activity did not increase significantly during erythroid differentiation until the reticulocyte stage. 4. The activity of four enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, adenosine deaminase and nucleoside phosphorylase, exhibited a similar pattern of change during erythroid differentiation. In the dividing cell compartment their activity was relatively high but exhibited a steep decline between the polychromatic stage and the orthochromatic stage, that is, as the cell completed its final cell division and moved from the dividing to the non-dividing compartment. After this the activity of these enzymes was stabilized at a relatively low value, and this activity persisted at such a value until the reticulocyte stage. 5. Lactate dehydrogenase activity also declined after the cell had crossed from the dividing into the non-dividing stage, but in this case the decline was less than in the case of the above four enzymes. 6. Adenylate kinase activity was relatively constant in the dividing cell compartment but exhibited a 60 percent increase when the cell passed from the dividing into the non-dividing compartment. 7. The cessation of cell division appears to coincide with a set of complex biochemical changes.     

Riboflavin deficiency in cultured rat hepatoma cells: A model for studying the hepatic effects of riboflavin deficiency

Summary The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme. This work was supported by grants from the VA Medical Research Service, the Diabetes Association of Greater Cleveland, and the National Institutes of Health (HD25299), Bethesda, MD. Portions of the work presented here were presented at the 71st meeting of the Endocrine Society, Seattle, WA.     

Distribution and activity of certain dehydrogenases in the erythropoietic process in pigeons]

Cytochemical methods were applied for detecting of distribution and dynamics of dehydrogenase activity (H- and M-subunits of lactate dehydrogenase, malate and succinate dehydrogenase) during maturation of pigeon erythrocytes. In the erythroblasts the above enzymes were seen in the whole cell; in reticulocytes - only around the nucleus; in erythrocytes - on the border line between the nucleus and the cytoplasm. The cytophotometric data show a decrease in enzymatic activity during maturation being more significant in the period from the erythroblast to reticulocyte development than from the reticulocyte to erythrocyte development.     

A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Glutamate dehydrogenase activity in developing soybean seed: Isolation and characterization of three forms of the enzyme

Three forms of glutamate dehydrogenase have been isolated from developing soybean seed. Their intracellular locations could not be determined directly because organelles and marker enzymes showed abnormal distribution on sucrose density gradient fractionation. By analogy with enzymes from other parts of the plant, glutamate dehydrogenase 2 was shown to be located in chloroplasts and glutamate dehydrogenase 3 in mitochondria. Glutamate dehydrogenase 1 could not be located in this way because it is found only in the seed. The three enzymes are similar in pH optima, molecular weight and substrate specificity with respect to 2-oxoglutarate and l-glutamate. The mitochondrial enzyme is specific for NAD⁺. The chloroplast enzyme shows low activity with NADP⁺ relative to NAD⁺ but uses NADPH readily in the aminating direction. Glutamate dehydrogenase 1 is active with both nucleotides and is the only form to show substantial deaminating activity with NADP⁺. Glutamate dehydrogenases 1 and 2 are activated and stabilized by glutathione and 2-mercaptoethanol whereas enzyme 3 is unaffected. No significant metabolic control of any of the enzymes could be detected. Malate, citrate, adenine nucleotides and long-chain fatty acyl CoA derivatives gave slight inhibition at high concentrations. Amino acids had no effect on activity. A possible role for the enzyme characteristic of the developing seed is discussed in relation to nutrient supply during the accumulation of reserve materials in the seed.