Proteins of bacterial membranes. Purification of NADH-dehydrogenase by electrofocusing]

A highly purified preparation of NADH dehydrogenase was isolated from bacteria M. lysodeikticus membranes. The purification procedure involved extraction of the enzyme complex from isolated membranes by EDTA, solubilization of the complex by non-ionogenic detergent (1% Triton X-100), chromatography on DEAE-cellulose and electrofocussing in the pH gradient 4-6. The isoelectric point of the preparation is at 4.5; its main component is a protein with m.w. of about 76.000.     

Glycophorins B and C from human erythrocyte membranes. Purification and sequence analysis

We have developed methods for the preparative purification of two sialoglycoproteins (glycophorins B and C) from human erythrocyte membranes by high-performance ion exchange and gel permeation chromatography in the presence of Triton X-100. Glycophorin B was obtained without any detectable contaminants, and glycophorin C exhibited a purity of about 90-95%. The amino acid sequence of the intramembranous domain (residues 36-71) of glycophorin B was determined and found to be similar to that of the hydrophobic region of the major sialoglycoprotein (glycophorin A). The amino acid sequence of the hydrophobic domain (residues 49-88) of glycophorin C, that was also determined, agreed completely with the structure recently deduced from cDNA sequencing.     

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre- extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat- denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat- denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X- 100 extraction of isolated D. discoideum membranes results in a Triton- insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.     

Photophosphorylation and related properties of reaggregated vesicles from spinach photosystem I particles.

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH+4 are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Identification of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases in ethylenediaminetetraacetate-treated P2 membrane fraction of monkey brain basal ganglia

Tyrosine phosphorylation of a 55- and 60-kDa protein was observed when EDTA-treated P2 membrane fraction from monkey basal ganglia was incubated with [gamma-32P]-ATP in the presence of Zn2+. Other metal ions were less effective in this phosphorylation. The effect of Zn2+ did not appear to be due to its inhibition of a tyrosine phosphatase. In the presence of Mg2+/Triton X-100 instead of Zn2+, phosphorylation on tyrosine residues of a 17-kDa protein and the external substrate poly(Glu, Tyr) 4:1 copolymer was observed. Both Mg2+ and Triton X-100 were essential for this and Zn2+ inhibited both of these phosphorylations. Convincing evidence for the existence of Zn2+-dependent and Mg2+/Triton X-100-dependent tyrosine protein kinases was obtained when the two kinases could be separated by extraction of the membranes by Triton X-100. The Zn2+-dependent phosphorylation was present exclusively in the Triton-solubilized supernatant whereas the Mg2+/Triton X-100-dependent phosphorylation was found associated with the Triton-insoluble membrane fractions. Externally added histone could also be phosphorylated on tyrosine residues in a Zn2+- or Mg2+/Triton X-100-dependent manner by the supernatant or membrane fraction, respectively.     

Photophosphorylation and related properties of reaggregated vesicles from spinach Photosystem I particles

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylalation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH⁺₄ are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.     

Electron spin resonance and biochemical studies of the interaction of the polyamine, spermine, with the skeletal network of proteins in human erythrocyte membranes

Spermine (N, N'-bis(aminopropyl)-1,4-butanediamine) is a polyamine thought to be important in several cell regulatory processes. Previous studies had shown that spermine prevented the lateral diffusion of transmembrane proteins in human erythrocyte ghosts (Schindler et al. (1980) Proc. Natl. Acad. Sci. USA 77, 1457-1461). In this paper, we present results of studies on the effect of spermine on erythrocyte membranes by employing electron spin resonance spin-labeling techniques in conjunction with spin labels specific for skeletal proteins, bilayer lipids or cell-surface sialic acid of the membrane and by employing SDS-polyacrylamide gel electrophoresis analysis of extracted spectrin and Triton shells. The major findings are: (1) spermine significantly decreases the segmental motion of protein spin-label binding sites (P less than 0.0001), which are predominantly on cytoskeletal proteins; (2) addition of spermine leads to a significant increase in the rotational motion of spin-labeled terminal sialic acid residues (P less than 0.001), most of which are located on glycophorin A, a result which may be secondarily caused by spermine-induced aggregation of cytoskeletal proteins and the cytoplasmic pole of this transmembrane sialoglycoprotein; (3) spermine completely inhibits the low-ionic strength extraction of spectrin, the major protein of the skeletal network which is attached to the bilayer proteins by two or more connecting proteins; (4) pretreatment of ghosts with spermine followed by Triton extraction resulted in the retention of significantly increased amounts of Band 3 and other skeletal and bilayer proteins including Bands 4.2, 6 and 7 in Triton X-100 shells relative to that of control-treated ghosts. These results suggest that spermine acts both to increase protein-protein interactions in the cytoskeletal protein network and to bridge skeletal and bilayer proteins and are discussed with reference to possible molecular mechanisms by which spermine may influence cell functions.     

Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.     

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

Incorporation of a follicle stimulating hormone used for embryo transfer in cattle into multilamellar liposomes

A commercially available follicle stimulating hormone preparation (FSH-P) was successfully incorporated into multilamellar vesicles (liposomes). Multilamellar liposomes were found to contain 9.39 +/- 1.14 mg FSH-P (n=4) per 100 mg phospholipid or approximately 19.0% of the original material used to form the liposomes. A 1% solution of Triton X-100 incubated with liposomes containing FSH-P for one-half hour at 37 degrees C released 33% of the entrapped FSH-P; more than 99% of the entrapped FSH-P was released when liposomes were incubated with a 2% solution of Triton X-100. Entrapment of FSH-P increased proportionally to the mole percentage of stearylamine used in liposome formation, suggesting that FSH-P is entrapped in the aqueous interstices of the cationic liposomes. Entrapment of FSH-P in stable liposomes suggests that these multilamellar vesicles may be useful as a FSH-P delivery vehicle used for the superovulation and embryo transfer of food animals.     

ISOLATION AND CHARACTERIZATION OF SOLUBLE ACIDIC LIPOPROTEINS FROM RAT BRAIN SYNAPTIC VESICLES

—Highly purified fractions of synaptic vesicles were prepared from rat cerebrum or cerebral cortex by density gradient centrifugation. Treatment of synaptic vesicle fractions by autoincubation, freeze-thawing and sonication in an isotonic alkaline-salt medium or in 0·1-0·3% (v/v) Triton X-100 released increasing quantities of synaptic vesicle protein and phospholipid into solution. When the soluble synaptic vesicle proteins were extracted with 0·1% (v/v) Triton X-100, the insoluble residue consisted mostly of 5–8 nm-thick membranes resembling the limiting membranes of intact synaptic vesicles. This finding, together with other considerations, suggested that the soluble proteins and accompanying phospholipids originated from the interior of the synaptic vesicles. A 0·3% (v/v) Triton X-100 extract of synaptic vesicle was fractionated by ultracentrifugal flotation and dialysis into three lipoprotein fractions: a low density lipoprotein (d < 1·21 g/ml), a high density lipoprotein (d = 1·21–1·35 g/ml) and a very high density lipoprotein (d > 1·35 g/ml). The phospholipid contents of the low, high and very high density lipoprotein fractions were 0·74, 0·38 and 0·20 mg/mg of protein, respectively. All three apolipoproteins had a high ratio of acidic to basic, and of polar to nonpolar, amino acids, and were rich in glycine, alanine and serine. Polyacrylamide gel electrophoresis of the alkaline-salt and Triton X-100 extracts of synaptic vesicles at pH 8·8 resolved a single anionic component which stained for protein, lipid (Sudan black B; iodine) and anionic groups (acridine orange). Polyacrylamide gel electrophoresis of synaptic vesicle extracts at pH 2·7 in 5 m urea and 0·25% (v/v) Triton X-100 resolved about 20 protein components. However, the protein profiles of electropherograms of the Triton X-100 and alkaline-salt extracts differed in certain respects, suggesting that these media to some extent solubilized different proteins. However, most of the protein bands in electropherograms of the Triton X-100 and alkaline-salt extracts also stained for lipid and anionic groups. In addition, two lipoprotein components in the alkaline-salt extract and four in the Triton X-100 extract contained carbohydrate. Isoelectric focusing of synaptic vesicle extracts resolved 6–8 protein fractions. The major fraction in Triton X-100 and alkaline-salt extracts had an apparent isoelectric point of approximately 4·2 and contained 0·24 mg of phospholipid per mg of protein. Soluble synaptic vesicle proteins released by incubating, freeze-thawing and sonicating in the alkaline-salt medium, and protein fractions of the latter obtained by electrofocusing had an absorption maximum of 260–265 nm which was enhanced in a cold 0·5 n perchloric acid extract, an observation suggesting the presence of a bound nucleotide. These findings demonstrate that rat brain synaptic vesicles contain a heterogenous array of soluble acidic lipoproteins which vary in buoyant density, lipid content, amino acid and carbohydrate composition and electrophoretic mobility in polyacrylamide gels. These acidic lipoproteins apparently comprise the bulk of the macromolecular contents of synaptic vesicles and probably serve as ‘carrier’ proteins for the binding and sequestration of the neurotransmitters.     

Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis.

A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.     

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.     

Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates

Treatment of synaptic membranes from rat brainstem and spinal cord with the nonionic detergent Triton X-100 at 1-10 microliters/mg protein caused a marked increase in glycine receptor (3H)strychnine binding expressed per mg of residual membrane protein. The effect was maximal (220 +/- 6% of control) at 5 microliters Triton/mg protein, while higher concentrations caused progressive loss of strychnine binding ability of membranes (27 +/- 6% at 25 microliters Triton/mg protein). The increase in strychnine binding caused by low Triton X-100 reflected an increase in membrane Bmax, the kD being unaffected by the treatment. The affinity of glycine analogues for receptor sites was not appreciably affected by the detergent either. The findings suggest an enrichment of the synaptic membrane preparation in glycine receptors, caused by the solubilization by Triton of membrane constituents not related to the receptor sites.     

The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate.

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). equilibrium dialysis gives an apparent binding constant of about 4 X 10(3) M(-1) at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH. Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 +/- 0.2, and located close to aromatic residues in glycophorin. Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.     

Cross-linking of the (Ca2+ + Mg2+)-ATPase protein.

The addition of cupric-1,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22 degrees C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the gel. At low temperatures (4 degrees C) dimer formation predominates even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane suspension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca2+ + Mg2+)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22 degrees C, freeze-etched membranes showed no alteration in the number or appearance of 80 A intramembranous particles. Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.