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a Department of Biology, University of California, Riverside, California 92521
b Department of Biology, University of Central Florida, Orlando, Florida 32816
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, gamete biology, signal transduction, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Membrane preparations have been used to characterize sperm proteins playing a role in sperm-egg interactions and ZP binding. For example, a fucosyltransferase was localized to organelle membranes of spermatogenic cells but moved to plasma membrane fractions during sperm maturation [5]. Fractionated sperm membranes were used to show that plasma membrane-enriched fractions inhibit the binding of live capacitated mouse sperm to intact ZPs [6]. Crude membrane fractions from mature mouse sperm contain heterotrimeric G-proteins that are activated in response to ZP3, suggesting that the membranes contain a ZP3 receptor (ZP3R) coupled to intracellular effectors [7]. This complex is retained in detergent-solubilized sperm membranes [8]. Porcine sperm membranes have been used to quantify high-affinity binding by the porcine ZP3/ZP1 complex [3], and detergent-solubilized porcine sperm membranes were used to identify ZP-binding proteins [9]. These previous studies demonstrated that sperm membrane fractions can be used to isolate proteins mediating sperm-ZP interactions.
To use membrane fractions to identify and characterize ZP-binding proteins, it is essential to understand the composition of the membrane fractions. In this study, we characterized membrane fractions recovered from a density fractionation protocol following either sonication (reported here) or vortexing [6] of mouse sperm. Our initial studies suggested that the yield of membranes was improved by using sonication rather than the vortex method to disrupt sperm. However, vortexing preferentially removed head membrane components [6], which could be useful for purifying ZP-binding proteins. To isolate proteins involved in sperm-ZP interactions during fertilization, the preparation of choice should effectively fractionate head membrane components. The distribution of membrane components in vortexed or sonicated membrane preparations was examined, and density fractionation of sonicated membrane vesicles achieved a differential distribution of head plasma membrane proteins. Thus, these membrane fractions are an effective tool for characterizing sperm proteins that mediate sperm-egg interactions, including primary and secondary ZP binding and acrosomal exocytosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Sodium 125I was purchased from New England Nuclear (Dupont, Boston, MA). Resins for gel filtration and chemicals for SDS-PAGE were purchased from Bio-Rad (Hercules, CA). Protease inhibitors, DNase I, hyaluronidase, BSA, Percoll (Pharmacia, Piscataway, NJ), and prestained markers for SDS-PAGE were purchased from Sigma (St. Louis, MO). Chloramine-T was purchased from Aldrich (Milwaukee, WI). Quantigold protein assay reagent was purchased from Diversified Biotech (Piscataway, NJ). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA).
Sperm Membrane Preparation
Sperm used for membrane preparations were not capacitated, consistent with previous work [6]. Cauda epididymal sperm were collected from 12- to 15-wk-old mice (ICR strain, Harlan Sprague-Dawley, San Diego, CA) in Hepes-buffered Whittingham buffer (HWB) [1]. All buffers contained a protease inhibitor cocktail of leupeptin (20 µg/ml), aprotinin (20 µg/ml), benzamidine (200 µg/ml), and PMSF (1 mM). Sperm were pelleted (100 x g, 15 min), HWB was removed, and sperm were resuspended in 1/10 TN (TN = 130 mM NaCl, 20 mM Tris, pH 7.4). Vortexed sperm membranes were prepared as previously described [6] by vortexing sperm suspensions for 2 min at setting 6 on a Vortex-Genie (Scientific Industries, Bohemia, NY). Sonicated sperm membranes were prepared with a probe sonicator (VirTis, Gardiner, NY) for 15 sec on ice repeated 3 times at intervals of 1 min. Cell debris was pelleted (500 x g, 15 min), the membrane supernatant was collected and diluted to 5 ml with 1/10 TN, and crude membranes (CMs) were pelleted by ultracentrifugation (Sorvall RC-60, SW 50.1 swinging bucket rotor, 108000 x g for 1 h at 4°C). CMs were fractionated on a sucrose step gradient (0.5-ml membranes, 1.5 ml 30% sucrose, 1.5 ml 40% sucrose, 1.0 ml 45% sucrose in TN) for 2 h at 125000 x g at 4°C. Membrane fractions were collected: band 1 = 0/30% interface, band 2 = 30/40% interface, band 3 = 40/45% interface, and band 4 = pellet. Protein content was quantified using Quantigold (Diversified Biotech, Piscataway, NJ), and the fractions were stored in liquid N2.
125I Labeling of Sperm Membranes
Sperm were collected and resuspended in HWB without BSA or added Ca2+ and containing 1 mM EGTA. Sperm were placed in an Iodogen-coated (Pierce, Rockford, IL) glass test tube for labeling. 125I (0.5 mCi) was added, and labeling was allowed to proceed for 30 sec with gentle swirling. Sperm were centrifuged through 0.5 ml of 10% sucrose in HWB (100 x g, 3 min). 125I-Surface-labeled sperm were resuspended in 1/10 TN, and membranes were prepared as described.
Enzyme Assays
Alkaline phosphatase (AP) activity was measured as previously described [10], using 10 mM p-nitrophenyl phosphate as the chromogenic substrate. Reactions were stopped at 10 min by addition of 0.5 M NaOH and the concentration of p-nitrophenol was determined by measuring the absorbance at 405 nm. The relative activity (nmol mg protein-1 min-1) of each of the 4 membrane bands was determined. Hyaluronidase activity was measured using the turbidometric method [11]. Assays were performed at pH 4, pH 5.3 (standard assay conditions), and at pH 7 to identify pH-sensitive isoforms [12]. Each assay was run for 12 h with 500-ng samples of membranes. Percentage transmittance (%T) was determined at 500 nm, and enzyme activity was expressed as normalized TRU per sample: TRU = (%Texp - %Tmin)/(%Tmax - %Tmin) [13].
Electron Microscopy
Sperm membrane fractions were prepared, and pellets of the samples were fixed in 3% paraformaldehyde, 3% glutaraldehyde, 0.1 M sodium cacodylate buffer, pH 7.4, for 60 min, washed in 0.1 M sodium cacodylate buffer, postfixed in 1% OsO4 in 0.1 M sodium cacodylate, washed with 0.1 M sodium cacodylate, and dehydrated in a graded series of acetone. Samples were infiltrated with acetone:Spurr resin (1:1) overnight, embedded in 100% Spurr for 4 h, transferred to fresh Spurr, and baked overnight at 70°C. Silver sections (
100 nm thickness) were stained with uranyl acetate and Reynold lead citrate, dried, and viewed on a Hitachi 500 transmission electron microscope at 75 kV at a total magnification of 30 000x.
Lectin Fluorescent Staining and Confocal Microscopy
Sperm were fixed with 4% formaldehyde in PBS for 10 min at room temperature (RT) and pelleted at 10 000 x g for 10 min to concentrate sperm. Sperm were resuspended in PBS, washed by pelleting again, and mounted on poly-L-lysine-coated slides by incubating a drop of fixed sperm on the slide in a humidified chamber for 30 min. Unbound sperm were removed by immersion in PBS 3 times for 5 min. Biotinylated lectins were added at a concentration of 100 µg/ml in 1% BSA in TBS (20 mM Tris, 150 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TTBS). Slides were incubated in a humidified chamber for 1 h and washed 3 times by immersion in PBS for 5 min. Localization of lectin was detected by avidin-fluorescein isothiocyanate (FITC) (2 µg/ml) in 1% BSA in TTBS for 1 h. Slides were washed as above, mounted in Gelmount (Biomeda, Foster City, CA), and viewed using a Zeiss confocal microscope. The fluorescent staining patterns observed with Lycopersicon esculentum lectin (LEA), Solanum tuberosum lectin (STA), or Arachis hypogaea lectin (PNA) were similar whether staining was performed on live or fixed sperm (unpublished results).
Electrophoresis, Immunoblotting, and Lectin Blotting
SDS-PAGE was performed using 8% reducing gels, except for sp56, which was run under nonreducing conditions. Molecular masses were estimated by comparison to prestained standards (Sigma). Proteins were blotted onto nitrocellulose [14], stained for total protein (Quantigold; Diversified Biotech), exposed to film for autoradiography (XRP-5; Kodak, Rochester, NY), or stained with antibodies or lectins. Equal amounts of protein were loaded for all samples. Autoradiography and immunoblots contained 500-ng samples, colloidal gold protein staining used 1-µg samples, and lectin blots used 2-µg samples. For gel autoradiography, 125I-labeled membrane fractions were electrophoresed and blotted onto nitrocellulose, and the blot was exposed to Kodak XRP-5 film. Immunoblots were blocked in TTBS as follows: TTBS/5% nonfat dry milk for 2 h at RT (sp56 and 
-tubulin), TTBS/5% fish gelatin overnight at 4°C (hexokinase, PH-20, ß-1,4-galactosyltransferase [GalTase]), or TTBS/3% BSA/1% ovalbumin, overnight at 4°C (antiphosphotyrosine PY-20). Blots were washed in TBS and incubated in primary antibody in blocking buffer. Blots were washed and incubated in secondary antibody conjugated to AP. Blots were washed and developed using 5-bromo-4-chloro-3-indolyphospate-p-toluidine (BCIP)/tetra-nitro-blue-tetrazolium chloride (Calbiochem) or BCIP/nitro-blue-tetrazolium chloride (NBT) (Pierce), which was diluted 1:20 in AP buffer (100 mM Tris, pH 9.5, 150 mM NaCl, 1 mM MgCl2), with the exception of PY-20, which was developed using enhanced chemiluminescence (Pierce). Antibody dilutions, incubation times, and temperatures were as follows: hexokinase: rabbit anti-hexokinase 1:1000, 2 h, RT; goat anti-rabbit-AP, 1:500, 2 h, RT; galactosyltransferase: rabbit anti-GalTase, 1:500, 12 h, 4°C; goat anti rabbit-AP, 1:250, 2 h, RT; mouse anti-PH-20, 1:1000, 12 h, 4°C; goat anti-rabbit-AP, 1:500, 2 h, RT; sp56: mouse anti-sp56 (7C5), 1:500, 2 h, RT; goat anti-mouse-AP, 1:500, 12 h, 4°C; tubulin: mouse anti--tubulin, 1:000, 2 h, RT; goat anti-mouse-AP, 1:500, 2 h, RT; phosphotyrosine: PY20, 1:2500, 1 h, RT; goat anti-mouse-AP, 1:10 000, 1 h, RT.
Lectin blots were blocked in TTBS/1% BSA for 2 h at RT. After three 5-min washes in TBS, blots were incubated with biotin-conjugated lectins at a concentration of 10 µg/ml in TTBS/1% BSA for 1 h at RT with shaking. Blots were washed with TBS, incubated with avidin-conjugated AP (2 µg/ml) in TTBS/1% BSA for 1 h at RT with shaking, washed 3 times in TBS, washed in AP buffer, and developed with BCIP/NBT (Pierce) diluted 1:20 in AP buffer. LEA recognizes GlcNAc (up to 3 nonconsecutive residues), PNA recognizes ßGal(1
3)GalNAc, and STA recognizes (GlcNAc)4. Specificity was verified by incubating parallel blots in 20 mM chitin/chitotriose (LEA), 100 mM chitin/chitotriose (STA), or 100 mM lactose (PNA). The molarity of the chitin/chitotriose was nominal; some material remained insoluble even after extensive stirring (i.e., overnight).
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RESULTS |
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Sonicated sperm CMs were fractionated on discontinuous sucrose gradients [5, 6, 15]. Four fractions were recovered and were designated band 1 through band 4. Transmission electron microscopy (TEM) of the sonicated membrane fractions revealed that band 1, band 2, and band 3 consisted entirely of membrane vesicles (Fig. 2). In contrast to the vortex preparation method, mitochondria were never observed in band 3. Band 4, the pellet, contained axonemal and midpiece fragments, and large segments of midpiece with dense fibers and mitochondria surrounding the axoneme were commonly seen. Occasionally, membrane fragments were detected along the axonemal fragments (Fig. 2). Rarely nuclei were seen, but membranes were not noted (data not shown). Thus, sonication is effective in removing membranes, and membrane fractions of 3 apparent densities can be recovered.
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We compared the protein composition of the sonicated membrane fractions with that of the vortexed membrane fractions [6]. First, samples from either sonicated or vortexed membranes were fractionated on sucrose step gradients. Equal amounts of protein from each fraction were separated by reducing SDS-PAGE, blotted onto nitrocellulose, and stained for protein using colloidal gold (Fig. 3). The sonicated preparations (Fig. 3A) and the vortexed preparations (Fig. 3B) showed a very similar overall protein distribution. For example, the p116 hexokinase is prominently represented in bands 1–3 of both preparations (stars). Bands 1 and 2 have a number of proteins in common, although these membrane fractions had distinct densities (0/30 vs. 30/40). Band 3 appeared to contain an array of proteins also present in bands 1 and 2 or in band 4, but some polypeptides were unique to this fraction. A number of prominent polypeptides segregated exclusively to band 4 and may represent axonemal or midpiece-associated proteins.
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One of the major differences between vortexed and sonicated membrane preparations is the relative amount of material recovered in each fraction using the 2 different preparative methods (Table 2). In comparison to the sonicated membrane preparations, the vortexed membrane preparations contained very little material in band 1 and band 4, as noted previously [6]. In the vortexed preparations, the majority of the protein (80%) was recovered in band 2. The relative amount of material recovered in band 3 was similar in both preparations. In contrast, the sonicated band 1 fraction contained 7.3-fold more material than was present in the vortexed band 1, with a corresponding decrease in the amount of material recovered in the sonicated band 2. However, when equal amounts of protein from each band were separated by SDS-PAGE and stained by colloidal gold, the membrane fractions from the vortexed and sonciated preparations appeared very similar (compare Fig. 3A with Fig. 3B).
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Although SDS-PAGE revealed differences in polypeptide enrichment among the bands, it did not reveal specific detail about the distribution of plasma membrane or head membrane proteins in the sonicated fractions. 125I surface labeling was used to identify enrichments in plasma membrane components (Fig. 4A) and immunoblotting (Fig. 4B and Table 3) to identify the distribution of known membrane proteins. To mark surface proteins, intact sperm were 125I labeled prior to sonication and fractionation of membrane vesicles. The 125I-labeled polypeptides were identified by gel autoradiography (Fig. 4A). Plasma membrane components segregated primarily to band 1 and band 2. Band 3 contained minor amounts of surface-labeled components, and band 4 contained only background levels (Fig. 4A). Labeling was performed at subsaturating conditions; thus only major cell surface components labeled. Therefore, the lack of 125I label on any given polypeptide should not be interpreted to mean that it is not a plasma membrane component. Cell surface labeling suggests that the majority of plasma membrane proteins fractionated to sonicated band 1, with lesser but significant amounts present in band 2 and band 3. These data are consistent with the TEM results (Fig. 2) showing that membrane components could be found in bands 1–3 but not in band 4.
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Membrane fractions were assayed for known sperm membrane antigens by immunoblotting and for enzymatic activities (Table 3). Immunoreactivity of the testis-specific hexokinase [16] is shown in Figure 4B. Immunoreactivity for each antigen tested was quantified by densitometry to evaluate the relative distribution in bands 1–4. Consistent with 125I surface labeling, the highest concentration of the plasma membrane markers hexokinase and GalTase were in bands 1 and 2. In contrast, phosphotyrosine staining that comigrated with the p116/hexokinase was approximately evenly distributed among bands 1–3. This finding suggests that the proteins in band 1 are relatively less phosphorylated than are those in bands 2 and 3. PH-20, which occurs on plasma and acrosomal membranes, was also found in highest concentrations in band 1 and band 2. Initial assays showed that hyaluronidase activity was highly enriched only in band 1 (Table 3). Because the plasma membrane and acrosomal forms have different pH optima [12] and the hyaluronidase assay is normally performed at an acidic pH, only some of the activity may have been detected. Membrane fractions were subsequently assayed for hyaluronidase activity at pH 4 and pH 7 (Fig. 5). The acidic activity was only prominent in band 1, but all fractions displayed high levels of neutral activity. The neutral activity was 6.6-, 7.6-, and 3.7-fold greater than that of the acidic hyaluronidase in bands 2–4, respectively, but neutral hyaluronidase activity was only 2.3-fold greater than acidic activity in band 1, suggesting a substantial enrichment of the acidic activity in the band 1 fraction. Alkaline phosphatase, a common enzymatic marker for membranes, had its highest activity in bands 1 and 4, suggesting both plasma membrane and either an axonemal or mitochondrial isoform of this enzyme.
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The distributions of specific sperm antigens were consistent with the 125I surface labeling, and there appeared to be some differential distribution of hyaluronidase activity. We used lectins to compare the distribution of proteins between the vortexed and the sonicated membrane preparations and to specifically correlate the distribution of membrane proteins with specific localization on the mouse sperm head. Membrane samples were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with lectins. Three lectins, PNA, STA, and LEA, illustrate the differential distribution of plasma membrane components in band 1 and band 2 (Fig. 6). In Table 4, lectin binding in the sonicated membrane fractions is compared with that in the vortexed membrane fraction, previously shown to be enriched in sperm head membranes [6], referred to here as head membranes. All polypeptides that stained prominently in the head membranes were also stained in the sonicated band 1–3 fractions. STA demonstrated differential distribution of proteins in bands 1 and 2.
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Fluorescent localization patterns that were obtained by lectin staining of intact sperm (Fig. 7) were different for PNA, STA, and LEA. Each of the lectins recognizes a different target oligosaccharide moiety. PNA stained the acrosomal crescent and entire tail of mouse sperm (Fig. 7A), although only the midpieces are shown. LEA stained the acrosomal crescent and posterior head (Fig. 7B), and STA stained the acrosomal crescent only (Fig. 7C). These lectins showed overlapping but distinct staining patterns on blots and together with the fluorescent localization pattern indicate that the sonicated membrane preparation achieved a differential fractionation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initially, we expected sonication to provide a greater yield of membranes than have previously used techniques. When sonicated membrane preparations were compared with vortexed preparations, the total amounts of membrane recovered were similar. Thus, sonication did not greatly enhance overall yield. It did, however, provide a different relative distribution of membrane vesicles. In the vortexed membrane preparations, virtually all of the membrane material was recovered in band 2. In contrast, sonicated membrane preparations had significant amounts of membrane material in band 1 and band 3.
In terms of starting material for isolation of sperm membrane proteins mediating sperm-egg interactions, the sonicated preparations have the advantage that the membrane vesicles are actually differentially distributed into 3 density fractions. Because the membranes derived by vortexing sediment almost exclusively in band 2, there is no real fractionation of membrane constituents. Thus, when testing the ZP-binding properties using sperm equivalents of membrane material [6] only band 2 had ZP-binding proteins; on a per sperm basis, there were limiting amounts of material in bands 1 and 3. In contrast, the sonicated membrane preparation provided sufficient material to test the bioactivity of each fraction, although isolation of polypeptides from band 3 could be technically challenging because this fraction contains the least amount of material.
When sonicated and vortexed preparations were compared on a protein mass basis, by using equal amounts of total protein from bands 1, 2, and 3 for SDS-PAGE, the overall protein composition appeared similar. These data suggest that the membrane preparations are similar but that the relative amount of vesicles of a particular density varies depending on the method of preparation. That is, vortexed preparations generate very little low-density (band 1) and high-density (band 3) vesicles, and virtually all the material is of intermediate density (band 2). In contrast, sonicating the membranes generated more of the low- and high-density vesicles, which suggests that sonication tends to generate smaller vesicles that would have only 1 class of microdomain (light, medium, or heavy). By comparison, vortexed sperm may result in larger vesicles containing multiple domains leading to a substantially higher number of vesicles of intermediate density.
The plasma membrane proteins generated by labeling with 125I prior to cell disruption were found in all the membrane-containing fractions. Band 1 was highly enriched for surface polypeptides in contrast to spermatogenic or somatic cells, which are mostly lipid. Band 2, which would correspond to plasma membrane vesicles in other cell types, contained many surface proteins but was not as enriched for surface components as was band 1. Band 3, which is highly enriched in organelle membranes in other cell types, contained significant amounts of plasma membrane proteins from mouse sperm. These results suggest that the sperm plasma membrane contains domains of both very high and very low density.
This observation is consistent with freeze fracture observations of mammalian sperm [17]. These studies have shown regions of densely packed intramembranous particles in various regions of the plasma membrane, including the acrosomal region of the head [17], which is the region of the sperm head where sperm first contact and adhere to the ZP [18] (unpublished results). Measurements of protein mobility in the sperm plasma membrane have also indicated the presence of membrane domains [19] and large lipid microdomains [20, 21]. A number of antigens have a very localized distribution on the sperm head [22]. From these studies, a picture of the mammalian sperm plasma membrane has emerged suggesting a highly polarized cell surface with well-demarcated lipid and protein domains.
The relative distribution of known sperm antigens, including several putative ZP receptors, in the sonicated membrane fractions was quantified by immunoblotting. The sperm surface GalTase, which has been implicated in both adhesion and induction of acrosomal exocytosis, was found primarily in band 1 and band 2 and in nearly equal amounts. Another putative ZP receptor, sp56 [4], which has been shown by immuno-TEM to reside within the acrosomal matrix [23], was localized primarily to band 1. PH-20, a glycosyl phosphatidylinositol-linked hyaluronidase that appears to play roles both in penetration of the cumulus layer and in ZP binding [24], occurs as 2 isoforms active at neutral and acidic pH optima, respectively. The localization of these isoforms of hyaluronidase showed that the neutral form is localized to both plasma and acrosomal membranes and that the acidic form is solubilized during the acrosome reaction [12]. When hyaluronidase activity of sonicated membrane fractions was measured at each pH, only band 1 was enriched in the acidic form, whereas bands 1–3 contained the neutral activity. The presence of sp56 and the acidic hyaluronidase in band 1 may indicate that some acrosomal components become encapsulated during the sonication procedure.
Lectins were also used as tools to observe the distribution of proteins in sonicated membrane fractions (bands 1–3) compared with the vortexed crude sperm membrane fraction, previously shown to be enriched in head membranes [16]. PNA stains the plasma membrane and/or acrosomal membranes of various mammalian sperm [25], LEA stains the plasma membrane and inner acrosomal membrane of boar sperm [26], and STA stains head plasma membranes of human sperm [27]. PNA and LEA stained polypeptides in the sonicated band 1 and band 2 fractions, whereas STA stained polypeptides from band 1. STA stained a number of polypeptides in band 1, and although the band 2 fraction contained similar amounts of 2 polypeptides (p116 and p32) as judged by the intensity of STA staining, band 2 did not stain at all for the other STA polypeptides that appeared in band 1, with the exception of the faint appearance of the 51-kDa polypeptide in some preparations. Fluorescent staining of sperm by STA was restricted to the acrosomal crescent. These data suggest that there is a differential distribution of sperm head plasma membrane proteins in band 1 and band 2.
These sonicated membrane preparations can be used to identify ZP-binding polypeptides with greater resolution than was possible in the previous study [6], in which only the band 2 membranes were able to compete for ZP binding. Thus, although there may be disadvantages to removing more than the head plasma membrane, these may be outweighed by the advantage of forming smaller vesicles containing more uniform domains and generating differential fractionation of head membranes in band 1 and band 2. Further, because virtually all of the membranes are removed and there is evidence to indicate that membrane domains are fractionated, these preparations may be useful for many types of studies of sperm function in addition to our particular interest of sperm-ZP interactions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was funded by National Institutes of Health grant HD 27244 and by grants from the University of California and the University of Central Florida. 
2 Correspondence: Catherine D. Thaler, Department of Biology, University of Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816. FAX: 407 823 5769; cthaler{at}pegasus.cc.ucf.edu
Accepted: August 13, 2001.
Received: March 8, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). B) Vortexed membrane fractions. The position of hexokinase is marked for comparison to the sonicated preparations. Molecular mass standards (kDa) are indicated to the left
