Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range

The effects of nonsaturating amounts (5–60 nmol/mg membrane protein) of $\text{p-}\text{chloromercuribenzoate}$ on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher $\text{p-}\text{chloromercuribenzoate}$ concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled $\text{p-}\text{chloromercuribenzoate}$. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to $\text{p-}\text{chloromercuribenzoate}$. Other reagents including $\text{N-}\text{ethylmaleimide}$, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K⁺-ATPase and free of any other enzyme marker other than K⁺-activated $\text{p-}\text{nitrophenyl}$ phosphatase.the 5′-nucleotidase and basal Mg²⁺-ATPase are clearly separated from the latter enzymes.Osmotic shock, Triton X-100 treatment or K⁺ ionophores increased the K⁺-ATPase activity in isotonic conditions, but $\text{K}{}^{\mathrm{+}}\text{-p-}\text{nitrophenyl}$ phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH₄⁺. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K⁺ is rate limiting for the ATPase activity but not for the $\text{p-}\text{nitrophenyl}$ phosphatase activity. It is concluded that K⁺ site for the ATPase is internal whereas the K⁺ site for the $\text{p-}\text{nitrophenyl}$ phosphatase is external, hence, the K⁺ site must be mobile across the membrane.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active $\text{p-}\text{aminohippurate}$ uptake in the rabbit kidney cortical slice preparation. Efflux of $\text{p-}\text{aminohippurate}$ from the slice was shown to be independent of external Na⁺ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing $\text{p-}\text{aminohippurate}$ increased intracellular concentrations of both Na⁺ and K⁺, and $\text{p-}\text{aminohippurate}$ accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and $\text{p-}\text{aminohippurate}$ resulted in a net increase in intracellular Na⁺ concentration but no $\text{p-}\text{aminohippurate}$ accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na⁺ concentrations and these directly reflected their respective $\text{p-}\text{aminohippurate}$ uptake. These results suggest that the Na⁺-gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na⁺ possibly has an intracellular role through its stimulation of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ channeled to energizing the $\text{p-}\text{aminohippurate}$ accumulative mechanism.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.     

The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca²⁺ pump of human red cell membranes catalyzes the hydrolysis of $\text{p-}\text{nitrophenyl}$ phosphate. The requirement for ATP of the Ca²⁺-$\text{p-}\text{nitrophenylphosphatase}$ activity was studied in relation to the two classes of site for ATP that are apparent during Ca²⁺ -ATPase activity. 2. (a) The $\text{K}{}_{0.5}$ for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ extrapolated at 0 mM PNPP is equal to the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the Ca²⁺ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca²⁺ -ATPase or as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$. 4. At ATP concentrations that almost saturate the high-affinity site, Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity increases as a function of PNPP along an S-shaped curve, while Ca²⁺ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca²⁺ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca²⁺ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca²⁺ -ATPase activity at the high-affinity size persists during Ca²⁺ -$\text{p-}\text{nitrophenylphosphatase}$ activity.