Detergent-solubilized Forms Research Articles

Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 μM) and chitohexaose (Kd = 17 ± 0.6 μM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

Interaction of bd-type quinol oxidase from Escherichia coli and carbon monoxide: Heme d binds CO with high affinity

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3-5 microM CO. The peak at 540 nm is probably either beta-band of the heme d-CO complex or part of its split alpha-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the gamma-band attributable to the reaction of the ligand with 10-15% of low-spin heme b558. High-spin heme b595 does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.

Resonance Raman spectroscopy of the cytochrome c oxidase from Paracoccus denitrificans.

Resonance Raman spectra are reported for the fully reduced unliganded and cyanide-bound mixed-valence forms of the cytochrome c oxidases from bovine heart and Paracoccus denitrificans in both detergent-solubilized forms and within their natural membrane environments. Comparison of the vibrational patterns observed for these enzymes indicates that overall the heme environments are similar for both. The only major differences seen between the spectra of these two enzymes are for vibrations associated with the low-spin bis(histidine)-coordinated heme cytochrome a. The data reported here serve to further illustrate the close structural and functional relationship between these evolutionarily distant enzymes. However, the data also demonstrate specific differences between the nature of the heme-protein interactions in the cytochrome a binding pocket which may be of mechanistic importance with regard to intramolecular electron transfer in these enzymes.

Fluoride is a slow, tight-binding inhibitor of the calcium ATPase of sarcoplasmic reticulum.

Addition of sodium fluoride in the millimolar concentration range to a solution containing the sarcoplasmic reticulum CaATPase undergoing turnover in its vesicular or nonionic detergent-solubilized forms produced a slow (time range of minutes) complete loss of enzymatic activity. In the presence of magnesium and the absence of calcium, similar results were obtained under nonturnover conditions. Time courses were adequately fit by a function corresponding to a monophasic transformation with a pseudo first order rate constant kobs. In the absence of Mg2+ (EDTA present) no inhibition developed; kobs depended hyperbolically on the Mg2+ concentration with the half maximal effect occurring near 4 mM. The fluoride concentration dependence of kobs showed no evidence of approaching saturation (highest [F-] used was 40 mM) and corresponded to a rate law which was approximately second-order with respect to fluoride. A number of ligands known to bind to the CaATPase were found to decrease kobs. Calcium prevented onset of fluoride inhibition with a midpoint in the micromolar range, implying an effect due to binding at the high affinity transport sites. ATP also protected with a midpoint in the micromolar range, consistent with an effect caused by active site binding of the nucleotide; protection was only partial, suggesting the ATPase can bind fluoride and ATP simultaneously. Prevention of fluoride inhibition by Pi occurred with a [Pi]1/2 of 12 mM at pH 6.5, a concentration similar to that which produces active site phosphorylation. Finally, protection by orthovanadate was found to be competitive and have a midpoint of 5 microM. These results point to an effect exerted at or near the phosphorylation site. The value of kobs increased from essentially zero above pH 8 to a plateau below pH 6; the transition had a midpoint near pH 7.2. Inhibition persisted after removal (with EGTA present) of unbound fluoride by dialysis. Reversal of fluoride inhibition was very slow, with a t1/2 of 16 h at 37 degrees C. These results suggest that fluoride behaves like a slow, tight-binding inhibitor of the ATPase and that the resulting complex is a stable transition (or intermediate) state analog. Plausible molecular bases for our results are that fluoride acts at the phosphorylation site as an analog of Pi or of hydroxide, which may be considered a substrate in the normal hydrolysis of the phosphorylated enzyme. A role for aluminum was ruled out after finding that the addition of EGTA to 10 mM or aluminum sulfate to 0.2 mM or deferoxamine to 0.5 mM produced no significant change in kobs.

Direct zinc binding to purified rhodopsin and disc membranes.

Using the radionuclide 65Zn, we have demonstrated the direct binding of zinc to purified rhodopsin. 65Zn is eluted with detergent-solubilized rhodopsin from concanavalin A columns and remains bound to the visual pigment through a subsequent gel-filtration step. Zinc binding to purified disc membranes is highly specific and, of the ions tested, copper is the best competitor. Equilibrium-dialysis experiments indicate that zinc binding to detergent-solubilized forms of rhodopsin may increase on bleaching the photopigment. These results may have important implications for studies that indicate that zinc plays a role in retinal degeneration and normal photoreceptor physiology.

Soluble forms of the rabbit adipose tissue and liver growth hormone receptors are antigenically identical, but the integral membrane forms differ

Cytosolic, detergent-solubilized and membrane-bound growth hormone (GH) receptors from rabbit adipose tissue and liver were tested for reactivity with a panel of monoclonal antibodies (MAbs). The cytosolic and detergent-solubilized forms of adipose tissue and liver GH receptors were identically reactive with four precipitating and two hormone-binding-site-directed MAbs. However, the membrane-bound form of the adipose receptor was 1000-fold less reactive with one binding-site-directed MAb (MAb 7) than the membrane-bound liver GH receptor. Reactivity with another inhibitory MAb (MAb 263) was identical for adipose tissue and liver membrane GH receptors. The relative potency of 22,000-Mr and 20,000-Mr forms of human GH was identical in assays with liver and adipose tissue membrane receptors. Thus, contrary to earlier suggestions, the discrepancy between the growth-promoting and insulin-like activities of 20,000-Mr human GH cannot be rationalized by a difference in the affinity of this hormone for 'somatogenic' and 'metabolic' receptors when the comparison is made in the same species. Cross-linking studies showed that the major GH-binding subunit of liver and adipose tissue GH receptors had the same Mr (54,000 +/- 5000, reduced). The ligand-binding subunits of liver and adipose tissue receptors are identical by several criteria, but one epitope on the adipose tissue receptor appears to be masked upon membrane insertion, possibly by close association with a tissue-specific component. Tissue specificity may be determined by association of a ubiquitous GH-binding subunit with tissue-specific membrane components, rather than by differences in amino acid sequence.

Topographic studies of microsomal and pure prostaglandin H synthase

Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [ acetyl- 14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme. These results suggest that the microsomal and the pure soluble forms of the synthase share a single exposed protease-sensitive region, and that proteolytic cleavage in this region results in loss of cyclooxygenase activity; binding of the heme prosthetic group changes the structure of this region so as to reduce protease sensitivity and thus preserve cyclooxygenase activity. The rest of the polypeptide chain in the microsomal synthase is arranged so as to maintain a tight association with the endoplasmic reticulum membrane even after cleavage occurs in the protease-sensitive region.

Effect of hydrostatic pressure on the mitochondrial ATP synthase.

The effects of hydrostatic pressure on three different preparations of mitochondrial H+-ATPase were investigated by studies of the hydrolytic activity, of the spectral shift and quantum yield of the intrinsic protein fluorescence, and of filtration chromatography. Both membrane-bound and detergent-solubilized forms of the mitochondrial F0-F1 complex were reversibly inactivated in the pressure range of 600-1800 bar, whereas with soluble F1-ATPase the inactivation was irreversible. Pressure inactivation of soluble F1-ATPase was facilitated by decreasing the protein concentration, indicating that dissociation is an important factor. In the presence of 30% glycerol, soluble F1-ATPase becomes inactivated by pressure in a reversible fashion, recovering the original activity. ATPase activity measured in an aqueous medium returns to the original values when incubated under high pressure in a glycerol-containing medium without substrate and is even enhanced when Mg-ATP is present. ATP hydrolysis returns to 80% of its original value in the case of the F0-F1 complex. Fluorescence studies under pressure revealed a red shift in the spectral distribution of the emission of tyrosine fluorescence of soluble F1-ATPase. A decrease in the quantum yield of intrinsic fluorescence was also observed upon subjection to pressure. The fluorescence intensity decreased monotonically as a function of pressure when the sample was in an aqueous medium, whereas it presented a biphasic behavior in a 30% glycerol medium. Gel filtration studies demonstrated that the hydrodynamic properties of the F1-ATPase are preserved if the enzyme is subjected to pressure in the presence of glycerol but they are modified when the same procedure is performed in an aqueous medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Liposomal sustained-release delivery systems for intravenous injection V. Biological disposition of liposome-entrapped lipophilic prodrug of 1-beta-D-arabinofuranosylcytosine.

A lipophilic prodrug of 1-β-D-arabinofuranosylcytosine (Ara-C), N4-[N-(cholesteryloxycarbonyl) glycyl]-Ara-C (COCG-Ara-C) was entrapped in unilamellar liposomes or solubilized in pH 7.4 phosphate-buffered saline with hydrogenated castor oil polyethylene glycol ether (HCO-60), and its biological disposition was studied in mice. Regardless of dosage form, COCG-Ara-C was accumulated in the liver after intravenous (i.v.) injection, but not in the lung or kidney. In contrast, the blood clearance and spleen distribution of COCG-Ara-C varied with dosage form: slow blood clearance and increased spleen levels of the prodrug were observed for the liposome-entrapped form relative to the detergent-solubilized form. Both COCG-Ara-C preparations successfully maintained the plasma levels of Ara-C compared with Ara-C aqueous solution. However, the plasma Ara-C concentration time profiles were markedly different between the preparations. After i.v. injection of liposome-entrapped COCG-Ara-C, plasma Ara-C levels increased slowly for the first 16h and subsequently decreased gradually. On the other hand, rapid appearance of Ara-C in the circulation and subsequently gradual elimination were observed after injection of the detergent-solubilized prodrug. The relative bioavailabilities obtained with respect to Ara-C aqueous solution were 592% and 129% for the liposome-entrapped and detergent-solubilized forms, respectively. The extremely high bioavailability of liposome-entrapped COCG-Ara-C could be explained in part by the finding that the liposome-entrapped prodrug reduced the plasma clearance of the parent drug Ara-C. These results indicate the potential utility of COCG-Ara-C-bearing liposomes as a sustained-release delivery system of Ara-C applied by i.v. injection.

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site.

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.

Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure.

The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen‐tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m‐NaCl, 5 mm‐EDTA, 1% Triton X‐100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X‐100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent‐free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris‐HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (&lt;15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex‐tracts, in a manner that depends on the ionic conditions. In Tris‐HCl buffer, G1 is converted into a well‐defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm‐Ca2‐ to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X‐100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.

Freeze-fracture study of water-soluble, standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca 2+-ATPase

Conventional freeze-fracture electron microscopy was used to study water-soluble proteins and different forms of Ca 2+-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was close to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca 2+-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particles sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca 2+-ATPase. The results can be interpreted on the assumption that the Ca 2+-ATPase molecule is elongated, with an overall length of about 110 Å and a width in its largest part of about 75 Å. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.

Comparison of the association and orientation of gamma-glutamyltranspeptidase in lecithin vesicles and in native membranes.

gamma-Glutamyltranspeptidase purified following solubilization with Triton X-100 can associate with single-layered [14C]lecithin vesicles. Enzyme activity and radiolabeled vesicles were shown to co-migrate during Sepharose 4B chromatography and isopycnic sucrose gradient centrifugation. The enzyme-vesicle complex exhibits a density corresponding to that of a single enzyme molecule bound to a single vesicle, gamma-Glutamyltranspeptidase purified following a solubilization with papain does not bind to vesicles. In addition, papain treatment of vesicles containing the Triton-purified transpeptidase results in the release of 95% of the transpeptidase activity without release of internally trapped [3H]sucrose. The released transpeptidase is chromatographically identical to the papain-purified transpeptidase. gamma-Glutamyltranspeptidase activity associated with both native membranes and with lecithin vesicles exhibits a temperature-induced transition in its energy of activation. In contrast, the proteolytic- and detergent-solubilized forms of the enzyme exhibit a single energy of activation over the entire temperature range. These results suggest that gamma-glutamyltranspeptidase binding to vesicles is due to a papain sensitive sequence of amino acids and that the enzyme.vesicle complex closely approximates the interaction and orientation of gamma-glutamyltranspeptidase with brush border membranes.

The Arrhenius plot behaviour of rat liver 5′-nucleotidase in different lipid environments

The plasma membrane enzyme 5′-nucleotidase (EC 3.1.3.5) was prepared from rat liver as a complex with sphingomyelin or in detergent-solubilized forms. Each preparation exhibited a K m and Arrhenius break temperature indistinguishable from that of 5′-nucleotidase in plasma membranes. Measurement of fatty acid profiles, cholesterol and phospholipid content however showed a very wide variation between these preparations. We conclude that the biphasic nature of the Arrhenius plot of 5′-nucleotidase may be a property of the enzyme rather than its lipid environment.

Milk fat globule membranes. Inhibition by sucrose of the alkaline phosphomonoesterase

Sucrose, a widely used agent in the preparation of membranes, inhibited the alkaline phosphomonoesterase of the milk fat globule membrane in both its membrane-bound and detergent-solubilized forms. The inhibition was kinetically competitive and reversible by dialysis. However, its mechanism was more complex than simple competition with substrate because: (a) sucrose induced the appearance of prolonged time-lags in the progress curves of the enzyme; (b) the extent of inhibition and of the time-lags depended on the age of the membrane preparation, the period of pre-exposure of the membranes to sucrose, and the temperature of pre-exposure. On the other hand the acid phosphomonoesterase and the phosphodiesterase activities also present in the membrane preparations were unaffected by the disaccharide.

Homogeneous cytochrome b5 from human erythrocytes

Homogeneous cytochrome b 5 has been isolated from large volumes of human erythrocytes by sequential chromatography on DEAE-cellulose, Amberlite CG-50, Bio-Gel P-60, and DEAE-Sephadex A-50. A molecular weight of 15,300 was determined by SDS disc gel electrophoresis. Trypsin converted the protein to a smaller hemepeptide which was indistinguishable from trypsin-cytochrome b 5 of human liver microsomes by disc gel electrophoresis. The data suggest that erythrocyte cytochrome b 5 has the same structure as a segment of liver microsomal cytochrome b 5 and is intermediate in size between the trypsin- and detergent-solubilized forms of the liver protein.