ATP-dependent proton translocation and quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of a cytochrome-deficient mutant of Escherichia coli,.

1. ATP-dependent proton translocation and ATP-dependent quenching of the fluorescence of 9-aminoacridine were measured in inside-out vesicles derived from a cytochrome-deficient mutant of Escherichia coli. 2. ATP-dependent quenching of fluorescence was inhibited by nigericin gramicidin, NH4Cl, and carbonylcyanide-m-chlorophenylhydrazone. Inhibition was also produced by the ATPase inhibitors N,N'-dicyclohexylcarbodimide (DCCD) and diphenyl phosphorazidate (DPA), and by the respiratory chain inhibitors piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and An2+. The inhibition of ATP-dependent fluorescence quenching by the ionophores, uncouplers, and respiratory chain inhibitors was not due to an effect on ATPase activity which was insensitive to these agents. 3. By use of the ATPase inhibitors DCCD and DPA, or by replacing ATP with GTP, ITP and CTP, a correlation between the ATPase activity and the rate of ATP-dependent membrane energization, as measured by fluorescence quenching, was obtained.     

Evidence for an interaction between the membrane protein of a paramyxovirus and actin.

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.     

Enteric viruses in renovated water in Manitoba

Using various concentration procedures (including the gauze pad method, ultrafiltration, and adsorption-elution), enteric viruses, mainly vaccine strains of polioviruses, were isolated from 61.8% of sewage, 20.5% of effluents, 3.0% of river water, and 6.7% of drinking water samples collected from a waterway system in Manitoba. The presence of polioviruses in the drinking water of the town utilizing this waterway was not correlated with an increase in the number of infectious diseases reported.     

Energetics of galactose,proline, and glutamine transport in a cytochrome-deficient mutant of salmonella typhimurium

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the β-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca²⁺, Mg²⁺-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.     

Purification and characterization of the inactive Ca2+, Mg2+-activated adenosine triphosphatase of the unc A- mutant Escherichia coli AN120.

Resealing of erythrocyte ghosts in the presence of 4.5 mm Ca²⁺ induces the segregation of small membrane vesicles with a very high phospholipid:protein ratio and a high lysolecithin content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the vesicles consist mainly of the high molecular spectrin peptides, the Ca²⁺-induced increase of band IIa (M_r 198,000) which is not extractable at low ionic strength, and a weak peptide band in the 72,000 M_r region. Ca²⁺ ghosts and vesicles show significant differences with regard to the specific activities of several membrane-associated enzymes. The segregated vesicles dispose of an efficient outwarddirected Ca²⁺-transport system.     

Proteolytic digestion and labelling studies of the organization of the proteins in the outer membrane of Escherichia coli.

The arrangement of the proteins in the outer membrane of Escherichia coli was examined by treating intact cells and isolated membrane preparations with fluorescamine and with pronase. Intact wild-type cells, or those of a mutant in which the core region of the lipopolysaccharide was absent, were equally resistant to pronase treatment. The protein components of isolated outer membrane preparations varied in their rate of digestion and labelling with fluorescamine. The N-terminal portion of protein B was removed by pronase to yield a fragment (protein Bp) still embedded in the membrane. Protein Bp was not significantly enriched in nonpolar amino acids, suggesting that protein B may not be held in the membrane primarily by hydrophobic interactions. This was confirmed by reconstitution experiments in which protein B could be reassociated with itself, without lipopolysaccharide or phospholipid, in the presence of divalent cation such that pronase digestion of the reassociated material gave protein Bp.