Covalent binding of 4-nitrobenzyl mercaptan S-sulfate to the sulfhydryl groups of hepatic cytosolic proteins and bovine serum albumin with mixed disulfide bond formation

4-Nitrobenzyl [35S]mercaptan S-sulfonic acid ([35S]NBM S-sulfate), a new type of reactive metabolite of the thiol [35S]NBM in rat liver cytosol fortified with 3'-phosphoadenosine 5'-phosphosulfate, bound rapidly and covalently at pH 7.4 and 37 degrees C to the sulfhydryl groups of rat liver cytosolic proteins with formation of disulfide bonds. From the radioactive proteins was isolated and identified the sole amino acid adduct, S-([35S]NBM)cysteine, after their acid hydrolysis under the anaerobic conditions. Bovine serum albumin (BSA), a model protein with a single SH group, also reacted readily with radioactive NBM S-sulfate to form a disulfide bond in stoichiometric manner. S-([35S]NBM)-cysteine was also isolated and identified as the sole amino acid adduct from the well-washed, radioactive BSA after the same anaerobic acid hydrolysis. A normal hepatic level of GSH not only retarded the BSA-NBM adduct formation completely, but also detached the radioactivity from BSA by the reduction of the disulfide bond with formation of [35S]NBM and its disulfide. Of twenty-one amino acids examined at pH 7.4 and 37 degrees C, only cysteine reacted with NBM S-sulfate and afforded S-(NBM)cysteine with concomitant formations of S-sulfocysteine, cystine, NBM, and its disulfide.     

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [(35)S]sulphate, heparin is the major radioactive product. However, [1-(14)C]glucosamine and d-[(14)C]glucose are incorporated largely into the glycoprotein. 4. The fate of [(35)S]sulphate-labelled and [1-(14)C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t((1/2)) of [(35)S]sulphate-labelled material in cells is 70hr. and that of [1-(14)C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [(35)S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1-(14)C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [(14)C]sialic acid being concentrated in the microsomal fraction.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.     

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.     

Paradoxical stimulation by amino acids of the degradation of [35S]methionine-labelled, short-lived protein in isolated rat hepatocytes

A complete amino acid mixture inhibited the degradation of long-lived and [14C]valine-labelled short-lived protein in isolated rat hepatocytes, but paradoxically stimulated the degradation of [35S]methionine-labelled short-lived protein. The stimulation persisted in the presence of autophagiclysosomal pathway inhibitors like 3-methyladenine and propylamine, indicating the existence of an hitherto unrecognized non-lysosomal degradation mechanism with selectivity towards methionine-rich proteins or peptide regions.     

Chloroplast membrane sidedness. Location of plastocyanin determined by chemical modifiers

Intact grana and stroma membranes (outer membrane absent) and detergent or sonication disrupted thylakoid membranes were treated with the hydrophilic covalent chemical modifiers [³⁵S]diazonium benzene sulfonic acid ([³⁵S]DABS) and [¹⁴C]glycine ethylester plus 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CDIS). Plastocyanin was purified using column chromatography followed by polyacrylamide gel electrophoresis and the incorporation of [³⁵S]DABS and [¹⁴C]glycine ethylester into plastocyanin was determined by slicing the gels and counting the radioactivity in the plastocyanin band. Plastocyanin isolated from thylakoids disrupted prior to chemical modification binds two to four times as much of either modifier than the plastocyanin isolated from intact chloroplasts. This ratio is five to ten times lower than the ratio expected for a component buried behind the permeability barrier of a membrane. The data suggest that plastocyanin is partially exposed at the external surface of the thylakoid membrane rather than being completely buried in, or behind, the lipo-protein membrane.     

Extracellular metabolism of glutathione accounts for its disappearance from the basolateral circulation of the kidney

Glutathione labeled in each of its amino acid residues, the corresponding free amino acids, and gamma-glutamyl-amino acids were used to evaluate their renal basolateral transport and metabolism at physiological levels of glutathione. Recovery of label in the venous outflow was compared to that of co-administered inulin after a single-pass in vivo infusion of rat kidney. Metabolites of glutathione and of its constituent amino acids were determined. No net basolateral transport of glutathione was detected; instead there was extensive breakdown of glutathione by the actions of basolateral gamma-glutamyl transpeptidase and dipeptidase. Glutamate and 5-oxoproline showed net basolateral uptake. Recoveries of 35S greater than those of inulin were found after perfusion of [35S]cysteine and [35S]glutathione suggesting rapid net tubular reabsorption of cyst(e)ine. Recovery of label from perfused [U-14C]glycine was equivalent to that of inulin consistent with little or no net flux. Co-administration of large amounts of unlabeled metabolites together with the labeled glutathiones led to label recoveries closer to those of inulin, consistent with competitive inhibition of labeled metabolite transport. Treatment of rats with an inhibitor of gamma-glutamyl transpeptidase decreased basolateral glutathione metabolism and thus indirectly decreased transport of labeled metabolites. No net basolateral transport of gamma-glutamyl-amino acids was detected. Significant amounts of label perfused as [Glu-U-14C]glutathione appeared in the gamma-glutamyl-amino acid fraction of the renal venous outflows, providing direct evidence that glutathione is used in vivo for the formation of gamma-glutamyl-amino acids.     

Effects of Inhibitors of Protein Synthesis on Transmembrane Auxin Transport in Cucurbita pepo L. Hypocotyl Segments

Pretreatment of 2?0 mm segments of etiolated zucchini (Cucurbitapepo L.) hypocotyl with cycloheximide (CH) or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide(MDMP) eliminated the stimulation by N-1-naphthylphthalamicacid (NPA) of net uptake of [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA),but had relatively little effect on the net uptake of IAA inthe absence of NPA. The efflux of [1-¹⁴C]IAA from preloadedsegments was not substantially affected by inhibitor pretreatmentin the absence of NPA, but CH pretreatment significantly inhibitedthe reduction of efflux caused by NPA. Pretreatment with CHor MDMP did not affect net uptake by segments of the pH probe[2-¹⁴C]5,5-dimethyl-oxazolidine-2,4-dione ([2-¹⁴C]DMO), or thenet uptake of [¹⁴C]-labelled 3-O-methylglucose ([¹⁴C]3-0-MeGlu),suggesting that neither inhibitor affected intracellular pHor the general function of proton symporters in the plasma membrane.Both compounds reduced the incorporation of label from [³⁵S]methionineinto trichloroacetic acid (TCA)-insoluble fractions of zucchinitissue, confirming their inhibitory effect on protein synthesis. The steady-state association of [³H]IAA with microsomal vesiclesprepared from zucchini hypocotyl tissue was enhanced by theinclusion of NPA in the uptake medium. The stimulation by NPAof [³H]IAA association with microsomes was substantially reducedwhen the tissue was pretreated with CH. However, CH pretreatmentdid not affect the level of high affinity NPA binding to themembranes indicating that treatments did not result in lossof NPA receptors. It is suggested that the auxin transport site on the effluxcarrier system and the receptor site for NPA may reside on separateproteins linked by a third, rapidly turned-over, transducingprotein. Key words: Auxin carriers, auxin efflux, Cucurbita pepo, phytotropin receptors     

PROTEIN METHYLATION IN RAT BRAIN IN VITRO

Abstract— Protein-methylation activity in various organs of the rat was studied with S-adenosyl-L-[methyl-¹⁴C]methionine ([methyl-¹⁴C](SAM) as methyl donor. Activity of the enzyme was highest in brain and lowest in liver. Histones comprised approximately 20 per cent of the total radioactivity incorporated, and lysine-rich histone was the most active. Analysis of amino acids of the methylated proteins of rat brain showed arginine to be the amino acid most extensively methylated, but some methylation occurred in lysine residues. An additional [methyl-¹⁴C]-labelled amino acid was found near histidine on the amino acid column chromatogram.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

The origin of the sulfur atom of thiamin

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.     

Origin of the thiazole ring of camalexin, a phytoalexin from Arabidopsis thaliana.

The principal phytoalexin that accumulates in Arabidopsis thaliana after infection by fungi or bacteria is 3-thiazol-2'-yl-indole (camalexin). Detached noninoculated leaves of Arabidopsis and leaves inoculated with the fungus Cochliobolus carbonum were fed [35S]cysteine (Cys) and [35S]methionine. Inoculated leaves incorporated more than a 200-fold greater amount of radioactivity from [35S]Cys into camalexin, as compared with noninoculated leaves. The amount of radioactivity from [35S]Cys that was incorporated into camalexin from inoculated Arabidopsis leaves was 10-fold greater than the amount of radioactivity that was incorporated into camalexin from [35S]methionine. Additional labeling experiments were performed to determine whether other atoms of Cys are incorporated into camalexin. [14C]Cys and [35S]Cys were incorporated into camalexin with approximately the same efficiency. Cys labeled either with deuterium (D3-Cys[2,3,3]) or 13C and 15N ([U-13C,15N]Cys) was also fed to inoculated leaves of Arabidopsis; camalexin was analyzed by mass spectroscopic analysis. The average ratio of molecular ion intensities of 203/200 for [U-13C,15N]Cys-labeled camalexin was 4.22, as compared with 0.607 for the average 203/200 ratio for unlabeled camalexin. The mass fragment-ion intensity ratios of 60/58 (thiazole ring ion fragment) and 143/142 were also higher for [U-13C,15N]Cys-labeled camalexin, as compared with unlabeled camalexin. The 59/58 and 201/200 ratios were higher for D3-Cys-labeled camalexin as compared with unlabeled camalexin. These data are consistent with the predicted formation of the thiazole ring of camalexin from Cys.     

Selective activation of cyclic AMP dependent protein kinase by calcitonin in a calcitonin secreting lung cancer cell line

When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by 90% with 7-chloro-4-nitro[14C]benzofurazan ([14C]Nbf-Cl) at pH 7.3 and then gel-filtered, 1.25 mols of [14C]Nbf-O-Tyr and less than 0.1 mol of Nbf-N-Lys were formed per mol of enzyme. After adjusting the pH of the gel-filtered, modified enzyme to 9.0 and incubating it for 14 hrs. at 23 degrees C to promote O----N migration, 0.68 mol of Nbf-N-Lys were formed per mol of enzyme while about 16% of the original activity reappeared. Isolation of the subunits after the O----N migration showed that 90% of the incorporated 14C was present in the beta subunit, which contained 0.21 mols of [14C]Nbf-N-Lys per mol. A tryptic peptide which contained the majority of the 14C incorporated into the beta subunit was isolated and subjected to automatic amino acid sequence analysis contained 38 residues. The amino acid sequence immediately around the lysine residue labeled with [14C]Nbf-, K*, was found to be: ...I-G-L-F-G-G-A-G-V-G-K*-T-V-L-I-G... .     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

Glutathione utilization by lactating bovine mammary secretory tissue in vitro.

gamma-Glutamyltransferase (D-glutamyl transpeptidase, EC 2.3.2.2) activity has been shown to be located predominantly on the extracellular surface of the plasma membrane of lactating bovine mammary cells. Radioactive label from both oxidized ([14C]-gamma-glutamyl) and reduced ([35S]cysteinyl) glutathione was taken up and incorporated into acid-precipitable proteins of mammary tissue. Uptake was shown to involve the transport of free amino acids, and incorporation was shown to involve the action of gamma-=glutamyltransferase. These results indicate that lactating mammary tissue utilizes the constituent amino acids of glutathione for milk-protein synthesis.     

Mode of antibacterial action by gramicidin S

To elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experiment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 microgram/ml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6 micrograms/ml, and the cells were temporarily saturated with GS at 10 micrograms/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by adsorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. coli increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.