Detergent Requirement Research Articles

Surface hydrophilicity promotes bacterial twitching motility.

The bacterial type IV pilus (T4P) is a critical virulence factor for many medically important pathogens, some of which are prioritized by the World Health Organization for their high levels of antibiotic resistance. The T4P is known to propel bacterial twitching motility, the analysis of which provides a convenient assay for T4P functionality. Here, we show that bile salts and other detergents augment the twitching of multiple bacterial pathogens. We identified the underlying mechanism as the alteration of surface hydrophilicity by detergents. Consequently, hydrophilic surfaces like those of glass or plasma-treated polystyrene promote bacterial twitching, bypassing the requirement for detergents. The implication is that surface properties, such as those of tissues and medical implants, significantly impact the functionality of bacterial T4P as a virulence determinant. This offers valuable insights for developing countermeasures against the colonization and infection by bacterial pathogens of critical importance to human health on a global scale.

In Situ Assembly of Transmembrane Proteins from Expressed and Synthetic Components in Giant Unilamellar Vesicles.

Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells.

Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell “SMALPome”, membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be “non-specific” than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Synergistic effect of ultrasonication and detergent on protein extraction from soymeal

Soymeal is among the most enriching vegetal protein sources and is produced as a by-product of soybean seed oil processing. In the present study, extraction of soymeal proteins was performed using four different methods such as water, solvent, alkaline, and detergent. The non-ionic detergent-based extraction provided promising results. To further improve the extraction efficiency of soymeal proteins, the detergent-based extraction was combined with ultrasonication. Influencing parameters such as detergent concentration, solute to solvent ratio, temperature, ultrasound power, and duty cycle were optimized. Ultrasound-assisted detergent-based extraction yielded 76.71 ± 0.9% of total proteins at 0.01% detergent concentration, 1:20 solute to solvent ratio, 50°C temperature, 75% duty cycle, and 180W. The extract was analyzed for L-phenylalanine (L-PHE) content to verify soymeal as its major source. Novelty Impact Statement The combined use of detergents and ultrasound-assisted extraction achieved an enhancement in the extraction efficiency of soymeal proteins. Triton X-100 helped in solubilizing oil and aided the release of proteins that were present in storage cells encapsulated with oil. In addition, the cavitation effect by ultrasonication resulted in cell disruption and improvement in protein mass transfer. Application of ultrasound demonstrated 1.58 times higher protein extraction and 50% less detergent requirement than the detergent-based stirring method.

A Mini Review on Superhydrophobic and Transparent Surfaces.

In recent years, self-cleaning and transparent surfaces have been widely studied for application on smart windows, solar panels, camera lenses, and other optoelectronic devices. The self-cleaning properties can possibly extend the lifetime of these products and decrease, even eliminate, the requirement of chemical detergents and high labor costs of cleaning. It can also promote the overall efficiency of outdoor optoelectronic devices (e. g. solar cell panels) since dirt accumulation and bacteria growth can be slowed down, even inhibited on such surfaces. In this mini review, the fundamentals and conditions that govern superhydrophobicity and transparency are introduced, followed by the discussion of roughness as the competing factor for superhydrophobicity and transparency. Representative examples of the surface design and fabrication are introduced and future perspectives are shared. This mini review can help the research community better understand such surfaces and further accelerate its development for innovative practical applications.

Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent.

Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforwardprocedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Solubilization of Lipid Membranes by Styrene-Maleic Acid Copolymers: Importance of Polymer Composition and PH

The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as tool in membrane research due to its ability to directly solubilize lipid membranes into nanodisc particles without the requirement of conventional detergents. Although many different variants of SMA are commercially available, so far mainly those with a styrene-to-maleic acid monomer ratio of 2:1 or 3:1 have been used for model membrane studies as well as for membrane protein extraction.Here, we present a systematic investigation of the effect of SMA composition on its solution properties as well as on its interaction with membranes. A particular focus was the effect of pH, which strongly affects the charge density of the polymer. Using model membranes of di-14:0 PC lipids, we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer.In addition, we used calorimetry and fluorescence methods to investigate the influence of SMA composition on the properties of lipids in nanodisc particles formed of different saturated PCs. We found that all polymers affect the gel-to-liquid crystalline phase transition of the lipids but that the degree of their influence depends on the styrene-to-maleic acid ratio.Altogether, the results show large differences in behavior between different SMA variants, with the one having a styrene-to-maleic acid ratio of 2:1 showing both the highest efficiency in membrane solubilization and the best preservation of lipid bilayer properties in nanodisc particles.

Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers

The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as a tool in membrane research, due to its ability to directly solubilize lipid membranes into nanodisk particles without the requirement of conventional detergents. Although many variants of SMA are commercially available, so far only SMA variants with a 2:1 and 3:1 styrene-to-maleic acid ratio have been used in lipid membrane studies. It is not known how SMA composition affects the solubilization behavior of SMA. Here, we systematically investigated the effect of varying the styrene/maleic acid on the properties of SMA in solution and on its interaction with membranes. Also the effect of pH was studied, because the proton concentration in the solution will affect the charge density and thereby may modulate the properties of the polymers. Using model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids at pH > pHagg, we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer. Furthermore, it was found that a collapsed conformation of the polymer is required to ensure efficient insertion into the lipid membrane and that efficient solubilization may be improved by a more homogenous distribution of the maleic acid monomer units along the polymer chain. Altogether, the results show large differences in behavior between the SMA variants tested in the various steps of solubilization. The main conclusion is that the variant with a 2:1 styrene-to-maleic acid ratio is the most efficient membrane solubilizer in a wide pH range.

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes.

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Functional Similarities between the Protein O-Mannosyltransferases Pmt4 from Bakers' Yeast and Human POMT1

Protein O-mannosylation is an essential post-translational modification. It is initiated in the endoplasmic reticulum by a family of protein O-mannosyltransferases that are conserved from yeast (PMTs) to human (POMTs). The degree of functional conservation between yeast and human protein O-mannosyltransferases is uncharacterized. In bakers' yeast, the main in vivo activities are due to heteromeric Pmt1-Pmt2 and homomeric Pmt4 complexes. Here we describe an enzymatic assay that allowed us to monitor Pmt4 activity in vitro. We demonstrate that detergent requirements and acceptor substrates of yeast Pmt4 are different from Pmt1-Pmt2, but resemble that of human POMTs. Furthermore, we mimicked two POMT1 amino acid exchanges (G76R and V428D) that result in severe congenital muscular dystrophies in humans, in yeast Pmt4 (I112R and I435D). In vivo and in vitro analyses showed that general features such as protein stability of the Pmt4 variants were not significantly affected, however, the mutants proved largely enzymatically inactive. Our results demonstrate functional and biochemical similarities between POMT1 and its orthologue from bakers' yeast Pmt4.

Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

A detergent-free strategy for the reconstitution of active enzyme complexes from native biological membranes into nanoscale discs

BackgroundThe reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.ResultsThe styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.ConclusionsWe report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.

The Styrene-Maleic Acid Copolymer Extracts Active Complexes from Native Biomembranes

Amphipathic polymers have been widely used to maintain the solubility of membrane proteins and complexes following detergent solubilization. However, their ability to extract proteins directly from lipid bilayers has remained largely unexplored. Here we show that a copolymer composed of styrene and maleic acid pendant groups (SMA) extracts proteins form native membranes and reconstitutes them into polymer-bound lipoprotein particles. First, we found that the SMA copolymer disrupted the membranes of intact mitochondria in a concentration-dependent and saturable manner. This was evidenced by the collapse of the transmembrane electric field of the inner mitochondrial membrane and by the solubilization of mitochondrial membrane proteins, both of which were mediated by the SMA copolymer in a manner similar to that mediated by nonionic detergents. Second, following incubation of the SMA copolymer with mitochondrial membranes and chromatographic separation, we observed by transmission electron microscopy that the resulting polymer-bound particles were a monodisperse population of discoids. The dimensions of these particles were similar to those previously reported for particles derived from liposomes or proteoliposomes of synthetic phospholipids (Lipodisqs®). Finally, using mitochondrial respiratory Complex IV (cytochrome c oxidase) as a model enzyme, we demonstrate that the SMA copolymer can extract even large, multicomponent complexes from native lipid bilayers and maintain them in a fully functional state amenable to solution-based biophysical studies. This novel approach to membrane protein reconstitution obviates the requirement for detergents and is therefore better suited to preserving native annular lipids and protein stability in comparison with traditional solubilization techniques.

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N(1-402)) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N(1-402) recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINE®12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N(1-402) in NDSB-256 was successfully used in pull-down and BIAcore™ protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N(1-402) and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called "cytosolic" tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N(1-402) should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

Preparation of homogenous oligosaccharide chains from glycosphingolipids

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II(3)NeuAcGgOse(4)Cer) and GbOse(4)Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.

Crystallization of a proteolyzed form of the horse pancreatic lipase-related protein 2: structural basis for the specific detergent requirement

Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystallization since, as shown here, this proteolyzed form of PLRP2 is only crystallized after specific detergent stabilization of this region. This has been performed by the hanging-drop vapour-diffusion method at 291 K and exclusively in the presence of N,N-dimethyldecylamine-beta-oxide (DDAO). However, most crystals (>95%) are highly twinned and diffract poorly (to approximately 7-5 A resolution). Diffraction-quality trigonal crystals have unit-cell parameters a = b = 128.4, c = 85.8 A and belong to space group P3(2)21. A 2.9 A native data set was collected at ESRF on beamline ID14-2 with an R(merge) of 12.7%. Preliminary structural analysis provides a structural basis for the specific roles of DDAO.

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans.

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

A continuous fluorescence displacement assay for phospholipase D.

Phospholipases D (PLD) are receiving increasing attention as important enzymes in lipid signalling pathways. Their biochemical function is to remove the headgroup (e.g. choline) from phospholipids by hydrolysis thus producing phosphatidic acid (PA). Primary alcohols can displace the active site water resulting in transphosphatidylation to produce phosphatidylalcohols [ 11. Whilst dioleoyl phosphatidylcholine (DOPC) is a poor substrate for human nonpancreatic secretory phospholipase 4 (hnpsPLAJ [2] the presence of either phosphatidic acid or phosphatidyl-alcohols can promote activity (papers in preparation). We report here on a novel adaptation of a fluorescence displacement assay, used to monitor fatty acid release, which makes use of activation of hnpsPLA, to indirectly monitor PLD activity. Phospholipases act by the process of interfacial catalysis which can be summarised by the following scheme: E*E* + S + E*S 4 E*P+ E* + P in which E* represents the activated form of the enzyme bound to the interface. The consequence of this mechanism is that if the enzyme does not bind productively to the interface (E*) it remains in the E-form and interfacial catalysis cannot proceed. Thii is the case for hnpsPLA, in the presence of vesicles composed of longchain phosphatidylcholines. The ability of membrane components to promote the E + E* step can therefore have significant effects on the apparent activity of hnpsPLA,. We have examined the effects of phosphatidylmethanol and phosphatidic acid, produced in situ by PLD from Sireptomyces chromofuscus, on the activity of hnpsPLA,. This enhancement of PLA, activity forms the basis of the assay described here. The assay system described does not require the separation of products h m substrates, as is the case with radiometric assays. This fluorescence displacement assay is continuous and sensitive, adaptable to mictrotitre plate format and may be suitable for high throughput screening systems. In the current format pure unlabelled DOPC vesicles were used as substrate. There is no requirement for detergents, indeed these may be deleterious due to activating effects on hnpsPL4. The indirect nature of the assay means that it is susceptible to factors that might alter the hnpsPLA, activity independent of the PLD activity, e.g. calcium concentrations. Assays performed here were carried out in 1 ml aliquots of assay cocktail containing 0.1 M Tris-HC1,O.l M NaCl, 1 mM CaCl,, pH 8.0, DOPC (50 pg/ml) and 1 pM dansyl undecanoic acid (DAUDA, Molecular Probes, USA). The phospholipid was present as small unilammelar vesicles produced either by alcohol injection or by sonication. Liver fatty acid binding protein (FABP) [3] was added to give a 1 : 1 ratio with the fluorescent probe. The complex so formed yields a high fluorescent signal which was monitored over approximately 1 minute. A second trace was recorded immediately on addition of 1.1 pg hnpsPLA, and the rate of decrease of fluorescence assessed. This rate is attributable to a very slow hydrolysis of DOPC by hnpsPLA, (see Figure. 1: blank) which has negligable effect on the composition of the vesicles. Having established the background rate PLD could then be added.The fluorescence signal is monitored immediately on addition of enzyme. Typical traces are depicted in Figure 1. The enhancement in the rate of decrease in fluorescence is proportional to the amount of PLD present (see Figure 2). The addition of PLD alone (in the absence of hnpsPLAJ does not result in a decrease in the fluorescence signal (result not shown). The assay can be performed in a variety of buffer systems including Hank's balanced salt solution. A variety of substrates including whole cells (results not shown) can be used in place of DOPC so long as hnpsPL4 remains predominantly in the E form and thereby expresses a negligible background rate. From Figure 2 it can be seen that the transphosphatidylation assay (i.e. in the presence of 1% v/v methanol) is more sensitive to PLD activity. This is in agreement with observations for the effect of phosphatidyl alcohols and phosphatidic acid on hnpsPL4 activity.

Surface localization of P34H an epididymal protein, during maturation, capacitation, and acrosome reaction of human spermatozoa.

During epididymal transit, spermatozoa acquire new surface antigens that are involved in the acquisition of their fertilizing ability. We have previously described a 34-kDa (P34H) human epididymal sperm protein that shows antigenic and functional homologies with the hamster P26h. P34H is localized on the acrosomal cap of human spermatozoa and has been proposed to be involved in the interaction with the zona pellucida. The aim of this study was to document the expression of P34H on the sperm surface during transit along the male and female genital tracts. Immunohistochemical techniques were performed on human testes and epididymides by means of an antiserum specific for P34H. No labelling was detected on those spermatozoa found within the seminiferous tubules or in the vasa efferentia. P34H first appeared in the caput epididymidis and was restricted to the acrosomal cap. Signal intensity then increased considerably from the proximal corpus to the cauda region of the epididymis. After ejaculation, the same pattern of P34H distribution was observed, but the intensity was much lower than that characterizing the cauda epididymal spermatozoa. Strong labeling was restored after incubation in B2 medium and was maximal after 5 h of capacitation. After acrosomal exocytosis induced by a Ca2+ ionophore, the percentage of P34H-labeled spermatozoa decreased proportionally to the number of acrosome-reacted spermatozoa as determined by Pisum sativum-fluorescein isothiocyanate (FITC) labeling. P34H appeared to be strongly anchored to the sperm plasma membrane during epididymal transit as indicated by the requirement for detergent to extract this surface antigen from ejaculated spermatozoa. This confirms the importance of P34H binding to the sperm plasma membrane during epididymal maturation. We have previously proposed that P34H is involved in sperm-zone pellucida interaction. The appearance and accumulation of P34H on the sperm plasma membrane during epididymal maturation, followed by its inaccessibility associated with ejaculation, its unmasking during capacitation, and finally its elimination after the acrosome reaction, are in agreement with te proposed function of this sperm antigen.

The Potential of Fluorinated Surfactants in Membrane Biochemistry

Detergents are important reagents in membrane biochemistry. Since each membrane system studied places different demands on the detergent in terms of desirous physicochemical properties, detergents new to biochemistry must continuously be sought. Ammonium perfluorooctanoate (APFO) was investigated, as representative of fluorinated surfactants, in terms of its suitability as a "biological detergent." It did not interfere with the Markwell modification of the Lowry procedure at detergent concentrations of up to 2% (w/v). Critical micellization concentration (cmc) values (0.013-0.0275 M) for this detergent were determined in a number of buffers of biological interest. It was demonstrated that the detergent can be removed by dialysis, albeit slowly. This slow removal may be particularly useful for reconstitution/crystallization studies. Solubilization studies on several membrane systems containing the proteins listed (the major protein of the membrane sector of the vacuolar H +-ATPase (16 kDa protein); photosystem II; equine herpes virus (EHV) envelope proteins) indicate that it is a potent solubilizing agent, likely to enhance the yield in cases where solubilization has already been demonstrated, and, in other cases, to solubilize proteins formerly recalcitrant to solubilization. The removal of APFO from solubilized 16-kDa protein by means of Extracti-Gel D resin as a means of exchanging detergents quickly and with a minimum requirement for second detergent was investigated.