Proteins of bacterial membranes. Purification of soluble ATPase from Acholeplasma laidlawii

A purified preparation of ATPase (factor F1) from the Acholeplasma laidlawii was obtained. The purification procedure included extraction of the enzyme complex from the isolated membranes by ultrasonication, chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The specific activity of the ATPase was increased 30-fold as compared to the original activity. The Km value for ATP hydrolysis was 7,4 . 10(-4) M. ADP competitively inhibited the enzyme (Ki = 2,0 . 10(-4) M). Ouabain (2,5 . 10(-4) M) and dicyclohexylcarbodiimide (1,0 . 10(-4) M) did not inhibit the ATPase activity. The enzyme was activated by Mg2+, but was inhibited by a combination of Na+ and K+. The enzyme is cold-labile, but can be stabilized by storage in buffer solutions, containing methanol, glycerol or lecithin.     

Chloride fluxes across Acholeplasma laidlawii membranes.

Chloride fluxes across the cytoplasmic membrane of Acholeplasma laidlawii were studied by using the chloride sensitive fluorescent dye, 6-methoxy-N-(sulfopropyl)quinolinium. Chloride was found to penetrate the membrane passively. Chloride flux was dependent upon the transmembrane electric potential.     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

Acylated proteins in Acholeplasma laidlawii.

The covalent modification of membrane proteins by long-chain fatty acids was determined in two strains of Acholeplasma laidlawii by one-dimensional gel electrophoresis of radiolabeled membranes. Of the more than 50 membrane polypeptides detected, approximately 30 were labeled with [3H]palmitate, whereas covalent binding of [3H]oleate to membrane proteins could not be demonstrated. We suggest that in these wall-less bacteria, membrane protein acylation with saturated fatty acids may serve to ensure the structural integrity of the membrane.     

Immunological properties of antibodies against Mg(2+)-ATPase from Acholeplasma laidlawii membranes.

In the present study, antibodies were raised against the Mg(2+)-ATPase and the immunological relationships between the enzyme and other ATPase from a variety of biological membranes were determined. The anti Mg(2+)-ATPase antiserum inhibited 85% of the enzyme activity from A. laidlawii membranes. We demonstrate a specific selectivity of Mg(2+)-ATPase antiserum for antigenic determinants of the A. laidlawii membranes. Immunoblot studies of A. laidlawii membrane peptides indicated labeling of five bands, 66KD, 49KD, 34KD, 26KD and 13KD, corresponding to five subunits of the ATPase in A. laidlawii membranes.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Adenylate energy charge in Acholeplasma laidlawii.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.     

Production of a T-Cell Clone Which Reacts with Membrane Proteins of Acholeplasma laidlawii

The role of cellular immunity in mycoplasma infection is not completely understood. In this study, we established mycoplasma-specific T-cell clones to evaluate cellular immunity in mycoplasma infection. We developed a T-cell clone (G-10) which was stimulated with Acholeplasma laidlawii. The T-cell clone G-10, CD4⁺ and T-cell receptor (TCR) αβ⁺ recognized the 42- and 65-kilodalton (kDa) membrane proteins of A. laidlawii and responded to A. hippikon. Hence, the application of mycoplasma-specific T cells such as G-10 in animal models may allow the assessment of cellular immune response to mycoplasma infection.     

Presence of protein constituents of the gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii.

Acholeplasma species have been reported to lack a functional phosphoenolpyruvate:sugar phosphotransferase system (PTS). We show here that Acholeplasma laidlawii possesses activities of enzyme I, HPr, HPr(ser) kinase, and HPr(ser-P) phosphatase but lacks detectable activities of enzymes II of the PTS. HPr from this organism was purified, and the regulatory properties of the kinase and phosphatase were characterized and shown to differ from those of previously studied bacteria. The results suggest the presence of an incomplete PTS in A. laidlawii which has the potential to function in a unique regulatory capacity.     

Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.