Adolph Abrams

Dio 9 and chlorhexidine: Inhibitors of membrane-bound ATPase and of cation transport in Streptococcus faecalis

1. 1. Dio 9, an antibiotic of unknown structure, and the synthetic bis-guanidine Chlorhexidine (1,6-di-4′-chlorophenyldiguanidohexane), inhibit the ATPase associated with isolated protoplast membranes of Streptococcus faecalis. They inhibit both the native, membrane-bound enzyme, and the solubilized protein. The effects of Dio 9 and Chlorhexidine differ in this respect from those of N,N′-dicyclohexylcarbodiimide which was previously found to inhibit only the membrane-bound ATPase. 2. 2. When added to intact cells, Dio 9 and Chlorhexidine inhibit the net uptake of K+ by exchange for Na+ and H+, and also the stimulation of glycolysis associated with K+ uptake. However, they do not inhibit autologous exchange of 42K+ for K+. 3. 3. Dio 9 and Chlorhexidine apparently do not inhibit the generation of ATP via glycolysis. Partial inhibition of glycolysis by these compounds may be a secondary consequence of the inhibition of ATPase. 4. 4. The finding that Dio 9 and Chlorhexidine inhibit both the ATPase and net K+ uptake by S. faecalis supports the hypothesis that the ATPase is involved in ion transport.

Role of Mg2+ ions in the subunit structure and membrane binding properties of bacterial energy transducing ATPase.

Abstract ATPase extracted from Streptococcus faecalis membranes was purified by preparative slab gel electrophoresis in the presence of Mg ++ (plus Mg 2+ ATPase) and without Mg 2+ (minus Mg 2+ ATPase). The subunit composition and membrane binding capacity of both preparations was then examined. The plus Mg 2+ ATPase had 5 types of subunits (αβγδϵ) and reattached normally to depleted membranes. The minus Mg 2+ ATPase had the αβγ and ϵ chains, but no δ chain, and failed to reattach to membranes. These data indicate that Mg 2+ or a similar cationic ligand anchors the δ chain to the core enzyme complex and that the δ chain in turn is needed for membrane attachment. For the plus Mg 2+ ATPase the data are consistent with the subunit stoichiometry and arrangement, ( α 3 β 3 γ ϵ)-Mg 2+ ) n −(δ).

RAPIDLY SYNTHESIZED RIBONUCLEIC ACID IN MEMBRANE GHOSTS FROM STREPTOCOCCUS FECALIS PROTOPLASTS.

Abstract 1. 1. Small amounts of RNA are present in washed membrane ghosts prepared by lysis of Streptococcus fecalis (ATCC No. 9790) protoplasts. Evidence was sought for differences between the membrane ghost RNA and RNA in other cell fractions, particularly ribosomes. 2. 2. After pulse labelling intact cells with [32P]orthophosphate, the membrane ghost RNA was found to be the most rapidly labelled as judged by measurement of the specific radioactivity of isolated 32P-labelled 2′(3′)-mononucleotides. Insoluble, easily sedimentable fragments of membrane ghosts obtained after sonication still retained RNA also with the most heavily 32P-labelled nucleotides. The nucleotide composition of the bulk and newly synthesized [32P]RNA in the membrane ghosts resembled the composition of ribosome RNA and the RNA in a DNA-rich fraction, but not s-RNA. 3. 3. Electron micrographs of the membrane ghosts indicate the presence of ribosomes. They are associated with a filamentous network and they are also situated in large numbers within a hollow tubular structure. 4. 4. It is concluded that in Streptococcus fecalis a rapidly synthesized RNA is associated specifically with membranous structures probably in membrane-associated ribosomes. 5. 5. Some information on the nature and amounts of other chemical components in the membrane ghosts is presented.

Selective penetration of ammonia and alkylamines into Streptococcus fecalis and their effect of glycolysis

Abstract Evidence was presented for the selective penetration of free base into Streptococcus fecalis cells suspended in solutions of NH 4 Cl, CH 3 NH 3 Cl, (CH 3 ) 2 NH 2 Cl and (CH 3 ) 3 NHCl at pH 7.0 or below. The selective penetration was measured by automatic continuous titration of the appearance of H + and was found to be practically instantaneous. Selective penetration of NH 3 was freely reversible and led to passive accumulation of NH 4 + . A mathematical equation was developed which satisfactorily describes the observed relation between amine uptake and extracellular amine concentration at constant pH. This equation permits an estimation of the intracellular pH and the rise in intracellular pH following penetration of free amine. Tentatively the intracellular pH of S. fecalis cells obtained from stationary growth phase is estimated to be about 5.0. Glycolysis in “aged” cells is retarded but is restored immediately following selective penetration of NH 3 or alkylamines at constant extracellular pH. K + and Na + also restored glycolysis but their action was slower. K + and Na + were found to elicit an efflux of H + at constant extracellular pH from non-glycolyzing cells possibly by ion exchange. It is concluded from these findings and a number of others as well that glycolysis in “aged” cells is inhibited by their low intracellular pH and is restored when the intracellular pH is raised.

Inhibitory action of carbodiimides on bacterial membrane ATPase

Abstract Dicyclohexylcarbodiimide (DCCD) inhibits the membrane ATPase of Streptococcus fecalis indirectly through a reaction with another membrane component as yet unidentified. Whether or not DCCD reacts covalenly to produce this inhibition is also not known. We have now found that several other carbodiimide compounds differing greatly from DCCD in structure and water solubility are also inhibitory. However, the hydrophobic carbodiimides were more potent by many orders of magnitude. Since structurally diverse carbodiimides could inhibit the membrane-bound ATPase we conclude that the inhibition results from a covalent reaction. The relatively high potency of the hydrophobic carbodiimides indicates that the reactive site is located within a non-polar region of the membrane. It is suggested that hydrophobic carbodiimides such as DCCD, by partitioning in the lipid phase of the membrane can reach a local concentration sufficiently high to react covalently with a membrane protein functional group.

Tightly bound adenine nucleotide in bacterial membrane ATPase

Summary Earlier studies have shown that Streptococcus faecalis organisms incorporate [32p]-orthophosphate into membrane ATPase ( Abrams and Nolan, BBRC (1972) 48 , 982 ). We have now found that [14C]adenine as well as 32Pi is incorporated into the enzyme in vivo . The in vivo labelled ATPase, solubilized and then purified by zonal sedimentation, contained labelled ATP, ADP and Pi. Binding experiments in vitro showed that labelled ATP, ADP and Pi bind to the isolated solubilized ATPase at concentrations of 5 × 10−5M. The ATPase-ligand complexes formed in vitro like those formed in vivo were sufficiently stable to be isolated by zonal sedimentation and polyacrylamide gel electrophoresis. The stoichiometry of in vitro binding of the labelled compounds was about 1 mol ATP, 1 mol ADP and 0.1 mol Pi per mol of enzyme.

Increased membrane atpase and K+ transport rates in streptococcusfaecalis induced by K+ restriction during growth

Summary The membrane ATPase in Streptococcus faecalis increased 1.5 – 2.1 fold when K+ in the growth medium was lowered to levels insufficient for the maintenance of optimal growth. Two other membrane enzymes tested, p-nitrophenylphosphatase and NADH dehydrogenase, did not increase. Cells with the induced increase in ATPase also exhibited increased rates of glycolysis dependent 86Rb+ uptake and net K+ uptake. The findings support the view that the membrane ATPase in S. faecalis functions in the active transport of K+ and that the organism responds adaptively to lack of K+ during growth by producing additional ATPase.

12. Bacterial Membrane ATPase

Publisher Summary This chapter discusses the methods of isolation and purification of Streptococcus faecalis membrane adenosine triphosphatase (ATPase). In the method commonly used to isolate the membrane of S. faecalis one first converts the cells to protoplasts using lysozyme to digest the wall and either 0.4 M sucrose or glycylglycine for osmotic stabilization. The protoplasts so produced are readily lysed either by osmotic shock or “metabolic lysis” to yield membrane ghosts. The ATPase activity associated with the membrane ghosts accounts for approximately 85% of the total cellular ATPase activity. While the small amount of soluble ATPase in the cytoplasmic fraction has not been purified it is probably identical or very closely related to the membrane-bound ATPase. This conclusion is based on polyacrylamide gel electrophoresis analysis, which shows that the cytoplasmic ATPase and solubilized membrane ATPase, detected on the gel by staining for catalytic activity, have identical mobilities (Nu). Whether or not a cytoplasmic ATPase exists in situ is difficult to say, but if it does the interesting possibility arises that it may have a precursor product biosynthetic relationship to the membrane-bound ATPase. The chapter also discusses ATPases from Escherichia coli, Micrococcus lysodeikticus , Bacillus stearothermophilus, Bacillus megaterium, and Rhodopseudomonas spheroides.

[39] The isolation of bacterial membrane ATPase and nectin☆

Publisher Summary The ATPase localized in the plasma membrane of Streptococcus faecalis is believed to function in the energized uptake of K + ions and amino acids. As described in this chapter, the enzyme can be isolated as a soluble homogeneous protein on a fairly large scale by the procedure devised by Schnebli. This procedure is based on the smaller scale isolation methods developed earlier by Abrams s and Abrams and Baron. A notable feature of the isolation procedure is that the dissociation of the ATPase from the membrane is accomplished under very mild conditions and without the aid of such agents as detergents or ultrasound. To detach the membrane-bound enzyme, protoplast membrane ghosts are washed repeatedly, first with solutions containing high salt and Mg 2+ and then with solutions at low ionic strength containing no Mg 2+ . This series of washes eventually leads to a spontaneous abrupt release of the ATPase from the membranes along with some other membrane proteins.

Cellular incorporation of 32 P-orthophosphate into the membrane ATPase of Streptococcus faecalis.

Abstract We have found that Streptococcus faecalis cells incorporate 32Pi into their plasma membrane ATPase. The labeled ATPase was isolated from cells grown in a 32Pi containing medium and also from non-growing cells suspended in 32Pi and glucose. To demonstrate that the enzyme contained 32P it was solubilized and then purified by chromatography on DEAE-cellulose, rate-zonal sedimentation and polyacry lamide elctrophoresis. Since the 32P remained firmly fixed to the catalytically active enzyme we conclude that it contains phosphate as a structural constituent. We estimate that one molecule of the ATPase contains 5 to 10 atoms of P.