Inhibitors Of Energy Production Research Articles

Abstract 2076: Inhibiting glycolysis and HIF1α: a novel approach to cancer therapy.

Abstract Cancer cells satisfy their increased need for energy under conditions of normal oxygen tension by undergoing a high rate of glycolysis followed by lactic acid fermentation, a process known as the Warburg Effect, rather than oxidative phosphorylation as observed in normal cells. Unlike proliferating cancer cells, the internal environment of most solid tumors is hypoxic due to an inadequate or disorganized blood supply. This results in tumors generating ATP through anaerobic respiration, a process many times less efficient than oxidative phosphorylation. The importance of anaerobic glycolysis for cancer cell energy generation makes it an attractive target for inhibiting cancer growth. However, agents currently in use for regulating energy production in tumors are relatively ineffective analogs of glucose or intermediates of the glycolytic pathway. We performed a high throughput siRNA screen using an siRNA library targeting all known open reading frames of the human genome (Dharmacon Inc.) to identify genes that when inhibited would block hypoxia inducible factor-1 alpha (HIF-1α) transcription factor luciferase reporter activity. A significant number of genes selected and validated were members of the glycolysis pathway. It is known that many glycolysis enzymes are induced by HIF-1α providing a feed forward loop between glycolysis and HIF-1α. We have addressed two questions: (1) what is the biological mechanism by which these two signaling systems regulate one another?; and (2) can specific inhibitors of glycolysis be developed with the added effect of inhibiting HIF-1α? Our mechanistic studies have shown that of the 40 enzymes of glycolysis involving 11 enzymatic steps and various enzyme isoforms, siRNA knockdown of Aldolase A (ALDOA) produces the maximum inhibition of HIF-1α activity. Further work indicated that ALDOA siRNA inhibition of HIF-1α is mediated through AMPK, a sensor of low ATP levels in the cell, since siRNA knockdown of AMPK results in rescue of HIF-1α activity. The effect of ALDOA inhibition is not mediated through a reduction in HIF-1α protein suggesting that, contrary to the canonical AMPK pathway, mTOR and its regulation of translation is not involved. Instead, p300 is phosphorylated by AMPK thus inhibiting its function as a co-activator of HIF-1α. A structure based drug design approach was employed for lead identification and optimization using the crystal structure of ALDOA (4ALD) and GOLD virtual screening platform. Three compounds have been identified as having effects on the modulation of ALDOA activity in a protein based biochemical assay as well as inhibitory effects on glycolysis itself using the XF96 Analyzer from Seahorse Bioscience. Here, we present an overview of ALDOA target identification, its mechanism as a regulator of HIF-1α activity and virtual drug design methodologies used in the search for novel pharmacological probes which act as dual inhibitors of energy production and the HIF-1α survival pathway. Citation Format: Geoffrey V. Grandjean, John Kingston, John Kenneth Morrow, Shuxing Zhang, Garth Powis. Inhibiting glycolysis and HIF1α: a novel approach to cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2076. doi:10.1158/1538-7445.AM2013-2076

Absence of RIP140 Reveals a Pathway Regulating glut4-Dependent Glucose Uptake in Oxidative Skeletal Muscle through UCP1-Mediated Activation of AMPK

Skeletal muscle constitutes the major site of glucose uptake leading to increased removal of glucose from the circulation in response to insulin. Type 2 diabetes and obesity are often associated with insulin resistance that can be counteracted by exercise or the use of drugs increasing the relative proportion of oxidative fibers. RIP140 is a transcriptional coregulator with a central role in metabolic tissues and we tested the effect of modulating its level of expression on muscle glucose and lipid metabolism in two mice models. Here, we show that although RIP140 protein is expressed at the same level in both oxidative and glycolytic muscles, it inhibits both fatty acid and glucose utilization in a fiber-type dependent manner. In RIP140-null mice, fatty acid utilization increases in the extensor digitorum longus and this is associated with elevated expression of genes implicated in fatty acid binding and transport. In the RIP140-null soleus, depletion of RIP140 leads to increased GLUT4 trafficking and glucose uptake with no change in Akt activity. AMPK phosphorylation/activity is inhibited in the soleus of RIP140 transgenic mice and increased in RIP140-null soleus. This is associated with increased UCP1 expression and mitochondrial uncoupling revealing the existence of a signaling pathway controlling insulin-independent glucose uptake in the soleus of RIP140-null mice. In conclusion, our findings reinforce the participation of RIP140 in the maintenance of energy homeostasis by acting as an inhibitor of energy production and particularly point to RIP140 as a promising therapeutic target in the treatment of insulin resistance.

Effect of Phospholipase A2 Inhibitors on the Release of Arachidonic Acid and Cell Viability in Cold-Stored Hepatocytes

We investigated the effect of phospholipase A2 (PLA2) inhibitors on PLA2 activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [3H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA2 inhibitors. PLA2 activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA2 activity was also suppressed (58% of control) by trifluoperazine (50 μM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 μM) suppressed PLA2 activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA2 activity. Thus, the inhibitory activity of these agents appears unrelated to Ca–calmodulin–phospholipid interaction but to have an inhibitory effect on PLA2 activity. To study the effects of PLA2 inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs–Henseleit buffer for 2 h at 37°C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA2 inhibitors blocked PLA2 activity, there was no suppression of LDH release. PLA2 may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.

Aspiration of THP1 into a micropipette. Mechanical deformation of monocytic THP-1 cells: occurrence of two sequential phases with differential sensitivity to metabolic inhibitors

Blood leukocytes can exhibit extensive morphological changes during their passage through small capillary vessels. The human monocytic THP-1 cell line was used to explore the metabolic dependence of these changes in shape. Cells were aspirated into micropipettes for determination of the rate of protrusion formation. They were then released and the kinetics of morphological recovery was studied. Results were consistent with Evans’ model (Blood 64:1028, 1984) of a viscous liquid droplet surrounded by a tensile membrane. The estimated values of cytoplasmic viscosity and membrane tension were 162 Pa.s and 0.0142 mN/m respectively. The influence of metabolic inhibitors on cell mechanical behavior was then studied: results strongly suggested that deformation involved two sequential phases. The cell elongation rate measured during the first 30 s following the onset of aspiration was unaffected by azide, an inhibitor of energy production, and it was about doubled by cytochalasin D, a microfilament inhibitor, and colchicine, a microtubule inhibitor. However, during the following 2 min, deformation was almost abolished in cells treated with azide and cytochalasin D, whereas the protrusion of control cells exhibited an approximately threefold increase in length. It is concluded that, although cells seemed to deform as passive objects, active metabolic processes were required to allow extensive morphological changes triggered by external forces.

Absorption of platelet-activating factor acetylhydrolase by rat intestine.

Platelet-activating factor acetylhydrolase (PAF-AH) is a 43-kDa protein that catalyzes the degradation and inactivation of this potent phospholipid mediator. PAF plays an important role in the pathogenesis of necrotizing enterocolitis (NEC), and the elevation of the plasma PAF-AH activity may be beneficial in the prevention of this disease. The activity of PAF-AH was transferred from the mucosal to the serosal fluid in intestinal sacs from neonatal rats. Translocation was highest on day 15, decreased by day 21, and disappeared by day 24. Greater transport of the enzyme was noted in the neonatal jejunum compared with duodenum or ileum. PAF-AH absorption in 15-day-old rats was decreased by the addition of inhibitors of energy production and by low temperature. We have, therefore, concluded that the enzyme transport is an energy-dependent process. It is suggested that PAF-AH found in milk may prove to be beneficial in the prevention of NEC by its translocation across the intestinal mucosa. The absorption of macromolecules in the neonate, in addition to providing a passive immunity, may also serve a protective role by inactivating certain proinflammatory agents, such as PAF.

Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation.

Aspartokinase II from Bacillus subtilis was shown by immunochemical methods to be regulated by degradation in response to starvation of cells for various nutrients. Ammonium starvation induced the fastest aspartokinase II decline (t1/2 = 65 min), followed by amino acid starvation (t1/2 = 80 min) and glucose limitation (t1/2 = 120 min). Loss of enzyme activity was closely correlated with the disappearance of the alpha subunit; degradation of the beta subunit was somewhat delayed or slower under some conditions. Pulse-chase experiments demonstrated that aspartokinase II was stable during exponential growth; the synthesis of the enzyme rapidly declined in response to nutrient exhaustion. The degradation of aspartokinase II was interrupted by inhibitors of energy production and protein synthesis but was not changed in a mutant lacking a major intracellular protease. Mutants lacking a normal stringent response displayed only a slight decrease in the rate of aspartokinase II degradation, even though aspartate transcarbamylase was degraded more slowly in the same mutant cells. These results indicate that although energy-dependent degradation of biosynthetic enzymes is a general phenomenon in nutrient-starved B. subtilis cells, the degradation of specific enzymes probably involves different pathways.

Motility processes in Acantharia (protozoa) III. Calcium regulation of the contraction ‐ relaxation cycles of in vivo myonemes

The myonemes in the marine pelagic protozoa Acantharia are contractile organelles involved in buoyancy regulation. It was previously shown that they can perform three kinds of movement: rapid contraction, slow undulation and slow relaxation. They consist of a periodically striated bundle of 2–4 nm nonactin filaments that are twisted in pairs and shortened by a coiling mechanism. After permeabilization or demembranation, contraction and relaxation can still be performed by varying Ca2+ concentration and ATP is not needed.In the present paper, we have studied the role of Ca2+ and inhibitors of energy production in intact cells. Our data suggest that; (i) the in vivo rapid contraction subsequent to mechanical or electrical stimulation is triggered by Ca2+ influx across the cell membrane; (ii) the slow contraction that takes place during the undulating movement depends on Ca2+ release provided by internal calcium stores; (iii) the rapid contraction as well as the progressive shortening that occurs during the slow undulating movement are caused by Ca2+ binding to the myoneme filaments; (iv) ATP is not directly involved in the saturation by Ca2+ of Ca2+ sensitive sites located along the myoneme microstrands; (v) regulation of the movements of Ca2+ within the cytoplasm depends mainly upon the alternative pathway of ATP production; (vi) calmodulin is presumably involved in this regulation. A tentative cytophysiologic interpretation of the mechanism of contractility is proposed.

Uptake of cadmium into cells in culture∗

Cadmium has been recognized as pollutant of the environment for many years and numerous studies on its toxic effects have been carried out. Little, however, is known about its metabolic behaviour e.g. why the metal is accumulated so extremely rapidly into the organs of men and animals. Since the study of the individual metabolic steps is very difficult in vivo cell cultures may be used to obtain first indications of what happens in the whole animal. We used CHO cells in monolayer culture to study the conditions under which the uptake of cadmium occurs. From serumfree medium the metal is accumulated rapidly in the cells. The uptake is inhibited very strongly by the presence of serum or albumin. Accumulation occurs against a concentration gradient and is dependent on the incubation temperature. Below 10°C no cadmium uptake is seen. Several substances which are known to affect cell metabolism have been used to influence cadmium accumulation. Neither inhibitors of energy production nor microtubule or microfil...

Toxicology of microbial toxins

Abstract

Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells.

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.

Comparative toxicology of AC 217,300 in various species of insects

AC 217,300 was highly toxic by topical application to adult Musca domestica and Blattella germanica, and larvae of Spodoptera eridania, with LD 50 values in μg/g of 20, 19, and 61, respectively. It was relatively nontoxic to Diabrotica undecimpunctata howardi beetles and Heliothis virescens larvae with LD 50 values of 165 and >883. When incorporated into baits, AC 217,300 was still highly toxic to M. domestica, B. germanica, and S. eridania. A significant increase in toxicity to H. virescens was observed although the percentage mortality was only 45%. AC 217,300 was not effective against D. undecimpunctata with either mode of application. A comparison of toxicity with tissue residues indicated that AC 217,300 was highly toxic to M. domestica, B. germanica, H. virescens, and S. eridania if it was absorbed. However, D. undecimpunctata appeared to be much less sensitive to AC 217,300, as only 18% mortality was observed with corresponding tissue concentrations two to six times those producing 95–100% mortality in M. domestica and S. eridania. Exposure of M. domestica to an AC 217,300-treated bait for 3–6 hr was sufficient to cause 50–100% mortality after 2 days and 95–100% mortality by 7 days. With B. germanica, a 1- to 3-day exposure resulted in 75–90% control within 7 days, while exposure for 3–7 days gave 90–100% control. Environmental temperature had a pronounced effect on the toxicity of AC 217,300 to both M. domestica and B. germanica. A 20-fold increase in toxicity was observed when the environmental temperature was raised from 21 to 32°C. This observation, in concert with the symptoms of intoxication, support the hypothesis that AC 217,300 is an inhibitor of energy production. Approximately 85 and 87% of the total residue of AC 217,300 extracted from treated M. domestica and B. germanica, respectively, was parent material. Six minor metabolites were detectable, of which three were tentatively identified by cochromatography with authentic standards. None of the identified metabolites had any insecticidal activity in vivo, suggesting that AC 217,300 is the actual toxicant.

Studies on the regulation of the biosynthesis of glucose-containing oligosaccharide-lipids. Effect of energy deprivation.

Energy deprivation, induced in thyroid slices by incubation with an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone) or inhibitors of respiration (N2 or antimycin A), led to a disturbance in oligosaccharide-lipid metabolism which was characterized by a pronounced depletion of the glucosylated (Glc3Man9GlcNAc2) dolichyl pyrophosphoryl saccharide with an attendant accumulation of the Man9GlcNAc2 and, to a lesser extent, the Man8GlcNAc2 lipid-linked species. A concomitant decrease in the N-glycosylation of proteins was furthermore observed. The distribution of lipid-derived oligosaccharides observed by thin layer chromatographic separation was similar whether radiolabeling was achieved by a metabolic ([14C]glucose or [2-3H]mannose incubation) or chemical ([3H]NaBH4) procedure. The latter method proved useful for measuring the levels of individual oligosaccharide-lipids in unincubated tissue, and in this way it was found that unless thyroid was rapidly frozen or immediately immersed in oxygenated medium a marked decrease of glucose-containing oligosaccharide-lipids occurred which could, however, be reversed by a short incubation. The addition of glucose to the slice incubations did not prevent the Man8-9GlcNAc2 accumulation brought about by the inhibitors of energy production, nor did it alter the oligosaccharide-lipid pattern of uninhibited slices. The effect of the inhibitors on metabolically labeled liver slices was similar to that observed in thyroid. The size of the total chloroform/methanol/water (10:10:3)-extractable oligosaccharide-lipid pool (Glc3-Man9GlcNAc2 to Man5GlcNAc2) of thyroid remained quite constant (about 2 nmol/g) regardless of the energy state, and no substantial change in the level of smaller oligosaccharide-lipids, primarily represented by Man2GlcNAc2 (0.5 nmol/g) was evident. Moreover, no change in the total pool sizes was observed in puromycin-treated slices. The possible mechanisms by which energy deprivation leads to an accumulation of the glucose-free oligosaccharide-lipids are evaluated. While it is likely that a selective impairment of glucosylation of newly formed molecules occurs, it is also possible that an imbalance occurs in a postulated glucosyltransferase-glucosidase shuttle with a trapping of the oligosaccharide-lipid in its unglucosylated form.

Cultured hepatoma cells as a model system for studying insulin processing and biologic responsiveness.

Monolayer cultures of minimal deviation hepatoma cells (H4-II-E-C3') bound and degraded insulin specifically, the apparent Ki value for insulin inhibition of both processes being 1 x 10(-8) M, indicating that cell-bound 125I-insulin is the substrate for subsequent hormone degradation in these cells as in isolated hepatocytes.1 The time course of insulin binding to its receptor depended on hormone concentration and temperature. Degradation of insulin also depended highly on temperature, with little or no degradation occurring at less than 20 degrees C, a temperature below which a membrane-lipid phase transition may block homone translocation or uptake. The effects of various agents on the binding and degradation of 125I-insulin also were tested. Agents believed to inhibit intralysosomal degradation of various proteins also inhibited the degradation of 125I-insulin by H4 cells (chloroquine, ammonium chloride, procaine, and lidocaine); inhibitors of energy production (dinitrophenol, sodium cyanide) inhibited degradation; an agent which inhibits microtubule function (vinblastine) blocked insulin degradation; and methylamine, reported to prevent receptor aggregation,2 also interfered with insulin processing. These findings are consistent with a model for cellular insulin processing, comprising receptor binding, clustering of receptors, endocytotic uptake, intralysosomal degradation, and extracellular release of some degradation products. H4 cells were highly sensitive to insulin. The KE for a half-maximal response of hormone-stimulated incorporationof 14C-glucose into glycogen was 10(-11) M insulin, corresponding to less than 1% receptor occupancy. This response was also mimicked by concanavalin A at a concentration of 10 microgram/ml. Vinblastine and chloroquine both significantly inhibited insulin-stimulated glucose incorporation into glycogen without affecting basal levels. However, since these inhibitory effects were not relieved by addition of excess insulin, it seems unlikely that their action on glycogen synthesis was exerted only at the level of the generation of an active intermediate or degradation product from hormone-receptor complexes. The hormone-sensitive H4 cells thus provide a useful system for further studies examining the role of insulin-receptor uptake in hormone action, receptor regulation, and signal termination.

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.

Effects of antimicrotubule agents, potassium and inhibitors of energy production on hCG secretion.

The BeWo trophoblastic cell line was employed to assess the requirement for microtubules and cellular energy in human chorionic gonadotropin (hCG) secretion. In contrast to the general inhibitory effect of colchicine and vincristine on hormone secretion in systems involving exocytosis, wide concentration ranges of these antimicrotubule agents caused enhancement of hCG secretion. Similarly, cytochalasin B, an agent that interferes with microfilament function, doubled both basal hCG secretion, and secretion of hCG stimulated by 1 mM dibutyryl cyclic AMP plus 1 mM theophylline (dbT). Inhibitors of cellular energy production (2,4-dinitrophenol, malonate, azide) decreased both secreted and cellular levels of hormone. High concentrations of K+ gave no enhancement of basal or dbT-stimulated hCG secretion, nor any reduction of cellular hCG levels. These findings contrasted with observations of others in secretory systems involving exocytosis, in which high K+ potentiated basal or stimulated hormone release and depleted cellular stores of hormone. It was concluded that the process of hCG secretion in the malignant trophoblast is fundamentally different from the mechanism of protein hormone secretion in other tissues.

Exit transport of a cyclic nucleotide from mouse L-cells.

At a concentration of 30 mum, 1,4,5,6,8-pentazaacenaphthylene, 3-amino-1,5-dihydro-5-methyl-1-beta-D-[5-14C]ribofuranosyl (NSC-154020), a tricyclic, 7-deazapurine nucleoside (TCN) is rapidly taken up by cultured mouse L-cells and converted to intracellular TCN-monophosphate, but not further metabolized. The TCN-monophosphate is also excreted by the cells into the medium. It is released by a saturable process against a concentration gradient and the release is inhibited by various inhibitors of energy production. This inhibition correlates with a depletion of the cells of ATP. Thus TCN-monophosphate excretion probably involves an active transport system. This transport system is highly temperature-dependent (the Q10 falls between 3 and 4) and is inhibited by papaverine, theophylline, Persantin, Probenecid, phenethyl alcohol and p-chloromercuribenzoate, but not by 500 muM cyclic AMP, AMP, or adenosine. Significant amounts of various natural phosphorylated intermediates (AMP, ATP, UTP, UMP, UDP-hexoses, and phosphorylcholine) are not released from the cells under similar experimental conditions either in the absence or presence of 30 muM TCN.

Metabolic requirements for interactions between nuclear and cytoplasmic membranes in the repair of damaged Amoeba nuclei.

Amoeba nuclear envelopes were damaged using microsurgery, and metabolic requirements for the steps in their repair were studied, and my placing the cells in a solution containing one of several metabolic inhibitors. The first step in repair, the association of pieces of endoplasmic reticulum with holes in the nuclear membranes, appears to be a passive process since it was not affected by inhibitors of energy production, RNA synthesis, or protein synthesis. In contrast, fusion of pieces of endoplasmic reticulum with the nuclear membranes at the margins of the holes was blocked by KCN and dinitrophenol, indicating that membrane fusion requires energy derived from respiration, but RNA and protein synthesis inhibitors did not prevent fusion of pieces of endoplasmic reticulum with the nuclear membranes. The subsequent completion of repair and restoration of intact nuclear membranes was almost completely blocked by inhibitors of respiration, and it was reduced in the presence of actinomycin and emetine, suggesting that in addition to a requirement for energy, some later steps in the repair of the nuclear membranes require RNA and protein synthesis.

Fate of alpha-bungarotoxin bound to acetylcholine receptors of normal and denervated muscle.

In organ culture, alpha-[(125)I]bungarotoxin bound to extrajunctional receptors of denervated muscle is lost from the tissue at a more rapid rate than the toxin bound to the junctional receptors of normal muscle. The rapid loss of toxin from denervated muscle can be blocked by inhibitors of energy production and protein synthesis, and may reflect turnover of the toxin-receptor complex in the membrane.

Stability of induced alpha-glucosidase activity in the absence of inducer.

Yeast cells containing induced α-glucosidase activity lose 10% of the original activity per hour when placed in phosphate buffer – glucose medium. The presence of inducer has no effect on the rate of loss. Induced cells placed in complex medium plus glucose or glycerol show an initial rise in α-glucosidase activity, and then a slow decrease which is accelerated when growth stops. The loss of induced enzyme activity in buffer–glucose medium is inhibited by inhibitors of energy production or protein synthesis whether they are added immediately or 2 or 4 h after suspension of cells in the medium. The ability of the inhibitors to block the loss of induced enzyme activity is correlated with their ability to inhibit protein synthesis. Ammonium sulfate inhibits the loss of α-glucosidase activity after a lag but amino acid or purine–pyrimidine mixtures do not. The loss of induced enzyme activity in cell-free supernatant solutions is less than 2% of the original activity per hour at 30°.