Pattern of organotin inhibition of methanogenic bacteria

Seven organotin compounds and tin chloride were tested for their effects on the methanogenic bacteria Methanococcus thermolithotrophicus, Methanococcus deltae delta LH, and Methanosarcina barkeri 227. The methanogens were strongly inhibited by triethyltin, tripropyltin, and monophenyltin compounds, generally at concentrations below 0.05 mM. Less inhibition by tributyltin and diphenyltin was observed at levels below 0.1 mM, but complete inhibition was observed at a 1 mM concentration. Tin chloride inhibited all methanogens, with nearly complete inhibition at a 1 mM concentration. There was no inhibition by tetra-n-butyltin and triphenyltin compounds even at 2 mM, the highest concentration tested. The 50 and 100% inhibitory concentrations of all compounds were estimated; these values varied with both the compound tested and the bacterium tested. The 50% inhibitory concentration estimate generally decreased (i.e., giving a higher toxicity) as the total surface area of the alkyltin molecules decreased. These results differ considerably from those reported previously for aerobic microorganisms (G. Eng, E. J. Tierney, J. M. Bellama, and F. E. Brinckman, Appl. Organometallic Chem. 2:171-175, 1988), where a clear correlation between increasing total molecular surface area and increasing toxicity was documented with a variety of organisms. Using the same procedures as for the methanogens, we examined the effects of organotin compounds on Escherichia coli growing aerobically or anaerobically. The E. coli inhibition pattern clearly resembled that seen in the data of Eng et al., under both aerobic and anaerobic conditions.     

Anaerobic Transformation of Furfural by Methanococcus deltae (Delta)LH

Methanococcus deltae (Delta)LH was grown on H(inf2)-CO(inf2) in the presence of various concentrations of furfural. Furfural at higher concentrations, namely, 20 and 25 mM, inhibited growth of this organism. At concentration of 5 and 10 mM, no inhibition of growth was observed. The other methanogens in this study were not inhibited by 10 mM furfural. Among the methanogens tested, M. deltae was capable of transforming furfural, whereas Methanobacterium thermoautotrophicum Marburg, Methanosarcina barkeri 227, Methanococcus thermolithotrophicus, and Methanobrevibacter ruminantium lacked this capability. One hundred percent removal of furfural was observed within 48 h of incubation in M. deltae cultures. The end product observed during furfural metabolism was furfuryl alcohol. An almost stoichiometric amount of furfuryl alcohol was produced by M. deltae. This transformation is likely to be of value in the detoxification of furfural and in its ultimate conversion to methane and CO(inf2) by anaerobic digestion.     

Inhibition of marine biofouling by bacterial quorum sensing inhibitors

Seventy eight natural products from chemical libraries containing compounds from marine organisms (sponges, algae, fungi, tunicates and cyanobacteria) and terrestrial plants, were screened for the inhibition of bacterial quorum sensing (QS) using a reporter strain Chromobacterium violaceum CV017. About half of the natural products did not show any QS inhibition. Twenty four percent of the tested compounds inhibited QS of the reporter without causing toxicity. The QS inhibitory activities of the most potent and abundant compounds were further investigated using the LuxR-based reporter E. coli pSB401 and the LasR-based reporter E. coli pSB1075. Midpacamide and tenuazonic acid were toxic to the tested reporters. QS-dependent luminescence of the LasR-based reporter, which is normally induced by N-3-oxo-dodecanoyl-L-homoserine lactone, was reduced by demethoxy encecalin and hymenialdisin at concentrations >6.6 μM and 15 μM, respectively. Hymenialdisin, demethoxy encecalin, microcolins A and B and kojic acid inhibited responses of the LuxR-based reporter induced by N-3-oxo-hexanoyl-L-homoserine lactone at concentrations >0.2 μM, 2.2 μM, 1.5 μM, 15 μM and 36 μM, respectively. The ability to prevent microfouling by one of the compounds screened in this study (kojic acid; final concentrations 330 μM and 1 mM) was tested in a controlled mesocosm experiment. Kojic acid inhibited formation of microbial communities on glass slides, decreasing the densities of bacteria and diatoms in comparison with the control lacking kojic acid. The study suggests that natural products with QS inhibitory properties can be used for controlling biofouling communities.     

Chloride affects the interaction between tyrosyl-tRNA synthetase and tRNA

The physiological concentration of free magnesium in Escherichia coli cells is about 1 mM, and there is almost no chloride in the cell. When the aminoacylation of tRNA by tyrosyl-tRNA synthetase was assayed at 1 mM free Mg2+, chloride (and sulphate) ions inhibited the reaction but acetate at the same concentration (< 200 mM) was not inhibitory. When the magnesium concentration was increased to 10 mM there was almost no chloride inhibition any more. Chloride strengthened the PPi inhibition, the Ki(app)(PPi) values at 1 mM free Mg2+ were 140, 120, and 56 microM at 0, 50 and 150 mM KCl, respectively. Chloride weakened the AMP inhibition, the corresponding values for Ki(app)(AMP) were 0.35, 0.5, and 0.9 mM. The value of Km(app)(tRNA(Tyr)) was clearly increased by chloride, being 22, 37, 93, and 240 nM at 0, 50, 100, and 150 mM KCl, respectively. Best-fit analyses of the PPi inhibition, AMP inhibition and Km(app)(tRNA) assays were accomplished using total rate equations. The analysis showed that the only kinetic events which are obligatory to explain the chloride effects are a weakened binding of Mg2+ to the tRNA before the transfer reaction and a weakened binding of Mg2+ to the Tyr-tRNA-enzyme complex after the transfer reaction. The dissociation constants for the former were 0.11, 0.3, and 2.8 mM and for the latter 0.6, 2.5, and 13 mM at 0, 50 and 150 mM KCl, respectively. Mg2+ is required for the reactive conformation of tRNA in the transfer reaction but chloride weakens its formation. After the transfer reaction the dissociation of Mg2+ from the aa-tRNA-enzyme complex enhances the dissociation of the aa-tRNA from the enzyme. The kinetics and the chloride effect were similar in the tyrosyl-tRNA synthetases from both Bacillus stearothermophilus and E. coli.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Assimilatory reduction of sulfate and sulfite by methanogenic bacteria.

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Inhibition of Methanogenesis from Acetate in Granular Sludge by Long-Chain Fatty Acids

The effect of four saturated long-chain fatty acids (caprylic, capric, lauric, and myristic) and one unsaturated long-chain fatty acid (oleic) on the microbial formation of methane from acetate was investigated in batch anaerobic toxicity assays. The tests were carried out with granular sludge from an upflow anaerobic sludge bed reactor. In this sludge, Methanothrix spp. are the predominant acetoclastic methanogens. Lauric acid appeared to be the most versatile inhibitor: inhibition started at 1.6 mM, and at 4.3 mM the maximum specific acetoclastic methanogenic activity had been reduced to 50%. Caprylic acid appeared to be only slightly inhibitory. Oleic acid was almost as inhibitory as lauric acid. Although adsorption of the inhibitor on the cell wall might play an important role in the mechanism of inhibition, the inhibition was found to be correlated with concentration rather than with the amount per unit of biomass. In practical situations, as in anaerobic waste treatment processes, synergism can be expected to enhance the inhibition of methanogenesis. In the present research a background concentration of lauric acid below its MIC strongly enhanced the toxicity of capric acid and (to an even greater extent) myristic acid.     

Effect ofl-homocysteine and derivatives on the high-affinity uptake of taurine and GABA into synaptosomes and cultured neurons and astrocytes

The effect ofl-nomocysteine and selected derivatives on the high-affinity uptake of the inhibitory neuroeffectors, GABA and taurine, was investigated in synaptosomes, and in cultured neurons and astrocytes. High-affinity uptake of taurine into synaptosomes was inhibited most effectively byl-homocysteine,Dl-homocysteine and homocystine whereas neuronal uptake was unaffected by any of the compounds tested. The high affinity uptake of taurine into astrocytes was markedly inhibited byl-homocysteine,l-homocysteic acid andl-homocystine. High-affinity GABA uptake into astrocytes was notably inhibited byl-homocystine, none of the other compounds tested causing appreciable inhibition below a concentration of 5 mM. Neuronal and synaptosomal high-affinity uptake of GABA was not significantly affected by any of the test compounds at concentrations below 5 mM. The implication of these results to the study of the mechanism of homocysteine-induced seizures and their relevance to the genetic disorder homocystinuria is discussed.     

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli.

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.     

Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.     

Production of Ethane, Ethylene, and Acetylene from Halogenated Hydrocarbons by Methanogenic Bacteria

Several methanogenic bacteria were shown to produce ethane, ethylene, and acetylene when exposed to the halogenated hydrocarbons bromoethane, dibromo- or dichloroethane, and 1,2-dibromoethylene, respectively. They also produced ethylene when exposed to the coenzyme M analog and specific methanogenic inhibitor bromoethanesulfonic acid. The production of these gases from halogenated hydrocarbons has a variety of implications concerning microbial ecology, agriculture, and toxic waste treatment. All halogenated aliphatic compounds tested were inhibitory to methanogens. Methanococcus thermolithotrophicus, Methanococcus deltae, and Methanobacterium thermoautotrophicum ΔH and Marburg were completely inhibited by 7 μM 1,2-dibromoethane and, to various degrees, by 51 to 1,084 μM 1,2-dichloroethane, 1,2-dibromoethylene, 1,2-dichloroethylene, and trichloroethylene. In general, the brominated compounds were more inhibitory. The two Methanococcus species were fully inhibited by 1 μM bromoethanesulfonic acid, whereas both Methanobacterium strains were only partly inhibited by 2,124 μM. Coenzyme M protected cells from bromoethanesulfonic acid but not from any of the other inhibitors.     

Inhibition of acetohydroxy acid synthase by leucine

The enzymatic reaction of acetohydroxy acid synthase in crude extracts of Escherichia coli K-12 is inhibited by leucine. Inhibition is most pronounced at low pH values and is low at pH values higher than 8.0. Both isoenzymes of acetohydroxy acid synthase present in E. coli K-12 (isoenzyme I and isoenzyme III) are inhibited by leucine. Isoenzyme I, which is responsible for the majority of acetohydroxy acid synthase activity in E. coli K-12 at physiological pH, is inhibited almost completely by 30 mM leucine at pH 6.25-7.0 and is not affected at all at pH values higher than 8.4. Inhibition of isoenzyme I by leucine is a mixed noncompetitive process. Leucine inhibition of isoenzyme III is pH-independent and reaches only 40% at 30 mM leucine. The inhibition of acetohydroxy acid synthase by leucine at physiological pH, observed in vitro in this study, correlates with the idea that acetohydroxy acid synthase is a target for the toxicity of the abnormally high concentrations of leucine in E. coli K-12.     

Sensitivity of food pathogens to garlic (Allium sativum)

The inhibitory activity of garlic ( Allium sativum ) against Staphylococcus aureus, Salmonella typhi, Escherichia coli and Listeria monocytogenes was measured by the 'turbidity' method. Minimum inhibitory concentration (MIC) of garlic at 80% inhibition level was calculated for these bacteria. All bacterial pathogenic strains tested were inhibited by garlic; E. coli was most sensitive and Listeria monocytogenes was least sensitive. Therefore, garlic has potential for the preservation of processed foods.     

Analysis of an mRNA exhibiting anomalous translational specificity.

Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested.     

Synthetic Analogues of the Termite Trail-following Substance

The effect of tripropyltin chloride (TPT) on some functional reactions in E. coli was investigated. The inhibition on respiration and protein, DNA and RNA syntheses were examined in vivo. Oxygen consumption by E. coli cells was scarcely inhibited even at the concentration of 30 µg/ml TPT. The incorporations of ¹⁴C-labeled amino acids into protein fraction were inhibited. Especially, in the case of l-leucine, it was inhibited 60% even at 10 µg/ml TPT. Both incorporations of ¹⁴C-adenine into DNA and RNA fraction were inhibited 50–60% at 20 µg/ml TPT. RNA polymerase was prepared from E. coli cells and the effect of organotin compounds on the enzyme activity was examined. Organotin compounds inhibited the enzyme activity only at high concentrations (5-10mm). and dialkyltin chlorides which possess no antimicrobial action showed the inhibition more intensely than trialkyltin chlorides. The effect on membrane-bound ATPase was also examined in vitro. We found that TPT has high inhibitory action on membrane-bound ATPase. However, it slightly inhibited the activity of ATPase separated from membrane.     

Toxicity of 1-methyl-4-phenylpyridinium derivatives in Escherichia coli.

Several derivatives of 1-methyl-4-phenylpyridinium (MPP+), i.e., 1-methyl-4-(4'-nitrophenyl)pyridinium (1), 1-methyl-4-(4'-cyanophenyl)pyridinium (2), 1-methyl-4-(3'-nitrophenyl)pyridinium (3), 1-methyl-4-(4'-chlorophenyl)pyridinium (4), 1-methyl-4-(4'-acetamidophenyl)pyridinium (5), and 1-methyl-4-(4'-aminophenyl)pyridinium (6), were synthesized in order to compare their toxicity with that of paraquat (PQ2+) in Escherichia coli. Addition of compounds 1, 2, and 3 to aerobic E. coli cell suspensions caused extracellular ferricytochrome c reduction, which was inhibited by superoxide dismutase in the same manner as that in the case of PQ2+. The rate of the ferricytochrome c (cyt. c) reduction was in the order of PQ2+ greater than 1 greater than 2 greater than 3, which is the same as that of the redox potentials of these compounds. On the other hand, MPP+, 4, 5, and 6, which have more negative potentials, had no effect on the cyt. c reduction. Compound 1 inhibited the growth of E. coli under aerobic conditions, but not under anaerobic conditions. The results show that compound 1 can act as a mediator for production of superoxide (O2-.), which seriously injures E. coli cells. However, though compounds 2 and 3 catalyzed the production of O2-. in E. coli cells, their activity of O2-. production was much lower than that of compound 1 or PQ2+. Thus, compound 3 had no effect on growth or survival of E. coli at 1 mM, while compounds 2 and 4 had both bacteriostatic and bacteriocidal effects which were independent of dioxygen (O2). The results show that the toxic mechanism is different from that of compound 1. MPP+, 5, and 6 had no effect on growth of E. coli. This paper shows that compound 1 is a novel enhancer of intracellular superoxide production, though the mechanism of toxicity of compounds 2 and 4 is not clear yet. The results suggest that the redox potential is a crucial factor for manifestation of the activity.     

Inhibition of homoserine dehydrogenase I by L-serine in Escherichia coli

We have reported that a major cause of growth inhibition of Escherichia coli by L-serine is its inhibition of homoserine dehydrogenase I (HDH I), which is involved in the biosyntheses of threonine and isoleucine [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. However, Patte et al. reported that L-serine does not inhibit HDH I [Patte, J.-C., Truffa-Bachi, P., & Cohen, G.N. (1966) Biochim. Biophys. Acta 128, 426-439]. In studies on the reason for these discrepant results, we found that the concentration of K+ and the pH in the assay mixture strongly influenced the inhibitory effect of L-serine. L-Serine strongly inhibited the HDH I activities in both the forward and reverse reactions between aspartate semialdehyde and homoserine at a physiological K+ concentration (100 to 200 mM) and physiological pH (7.5) for E. coli cells. On the other hand, two well-known inhibitors of HDH I, L-threonine and L-cysteine, strongly inhibited the activity regardless of the K+ concentration and pH.     

Inhibitory effect of creosote and its main components on production of verotoxin of enterohaemorrhagic Escherichia coli O157:H7

The inhibitory effects of creosote and its main components, alpha-methoxyphenol and o-ethylphenol, on production of verotoxin by enterohaemorrhagic Escherichia coli O157 (VTEC E. coli) were investigated. The production of verotoxin by VTEC E. coli was inhibited by the administration of 0.001-0.1% of creosote or o-ethylphenol (final concentration). On the other hand, weak inhibition of production of verotoxin was observed with 0.1% alpha-methoxyphenol administration. As the inhibitory effects were obtained below Minimal Inhibitory Concentration (MIC) values, these test compounds exerted their effects at the active site of VTEC E. coli cells prior to their production of verotoxin. These findings suggest that pre-administration of creosote and its main components might prevent human intestinal food poisoning by VTEC E. coli and contribute to maintenance of public health.     

Potential inhibitors from wet oxidation of wheat straw and their effect on growth and ethanol production by Thermoanaerobacter mathranii

Alkaline wet oxidation (WO) (using water, 6.5 g/l sodium carbonate, and 12 bar oxygen at 195 degrees C) was used for pre-treating wheat straw (60 g/l), resulting in a hemicellulose-rich hydrolysate and a cellulose-rich solid fraction. The hydrolysate consisted of soluble hemicellulose (9 g/l), aliphatic carboxylic acids (6 g/l), phenols (0.27 g/l or 1.7 mM), and 2-furoic acid (0.007 g/l). The wet-oxidized wheat straw hydrolysate caused no inhibition of ethanol yield by the anaerobic thermophilic bacterium Thermoanaerobacter mathranii. Nine phenols and 2-furoic acid, identified to be present in the hydrolysate, were each tested in concentrations of 10-100x the concentration found in the hydrolysate for their effect on fermentation by T. mathranii. At 2 mM, these aromatic compounds were not inhibitory to growth or ethanol yield in T. mathranii. When the concentration of aromatics was increased to 10 mM, the fermentation was severely inhibited by the phenol aldehydes and to a lesser extent by the phenol ketones. By adding the same aromatic compounds to WO hydrolysate (10 mM), synergistic inhibitory effects of all tested compounds with hydrolysate components were shown. When the hydrolysate was concentrated three- and six-fold, growth and fermentation with T. mathranii were inhibited. At a six-fold hydrolysate concentration, the total concentration of phenolic monomers was 17 mM; hence aromatic monomers are an important co-factor in hydrolysate inhibition.     

Inhibitory effect of polycations on phosphorylation of glycogen synthase by glycogen synthase kinase 3

Several polycations were tested for their abilities to inhibit the activity of glycogen synthase kinase 3 (GSK-3). L-Polylysine was the most powerful inhibitor of GSK-3 with half-maximal inhibition of glycogen synthase phosphorylation occurring at approx. 100 nM. D-Polylysine and histone H1 were also inhibitory, but the concentration dependence was complex, and DL-polylysine was the least effective inhibitor. Spermine caused about 50% inhibition of GSK-3 at 0.7 mM and 70% inhibition at 4 mM. Inhibition of GSK-3 by L-polylysine could be blocked or reversed by heparin. A heat-stable polycation antagonist isolated from swine kidney cortex also blocked the inhibitory effect of L-polylysine on GSK-3 and blocked histone H1 stimulation of protein phosphatase 2A activity. Under the conditions tested, L-polylysine also inhibited GSK-3 catalyzed phosphorylation of type II regulatory subunit of cAMP-dependent protein kinase and a 63 kDa brain protein, but only slightly inhibited phosphorylation of inhibitor 2 or proteolytic fragments of glycogen synthase that contain site 3 (a + b + c). L-Polylysine at a concentration (200 nM) that caused nearly complete inhibition of GSK-3 stimulated casein kinase I and casein kinase II, but had virtually no effect on the catalytic subunit of cAMP-dependent protein kinase. These results suggest that polycations can be useful in controlling GSK-3 activity. Polycations have the potential to decrease the phosphorylation state of glycogen synthase at site 3, both by inhibiting GKS-3 as shown in this study and by stimulating the phosphatase reaction as shown previously (Pelech, S. and Cohen, P. (1985) Eur. J. Biochem. 148, 245-251).