Permeability properties of apical and basolateral membranes of the guinea pig caecal and colonic epithelia for short-chain fatty acids

Unidirectional fluxes of short-chain fatty acids (SCFA) indicated marked segmental differences in the permeability of apical and basolateral membranes. The aim of our study was to prove these differences in membrane permeability for a lipid-soluble substance and to understand the factors affecting these differences. Apical and basolateral membrane fractions from guinea pig caecal and colonic epithelia were isolated. Membrane compositions were determined and the permeability of membrane vesicles for the protonated SCFA was measured in a stopped-flow device. Native vesicles from apical membranes of the caecum and proximal colon have a much lower permeability than the corresponding vesicles from the basolateral membranes. For the distal colon, membrane permeabilities of native apical and basolateral vesicles are similar. In vesicles prepared from lipid extracts, the permeabilities for the protonated SCFA are negatively correlated to cholesterol content, whereas no such correlation was observed in native vesicles. Our findings confirm that the apical membrane in the caecum and proximal colon of guinea pig is an effective barrier against a rapid diffusion of small lipid-soluble substances such as SCFAH. Besides cholesterol and membrane proteins, there are further factors that contribute to this barrier property.     

Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea Pig

In adapting several methods of membrane isolation we established a successful way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texas Red. Apical and basolateral enterocyte membranes were enriched 10- to 12-fold by differential precipitation and via a continuous sorbitol gradient. The membrane fractions were examined with regard to their phospholipid (PL) and fatty acid patterns and to their cholesterol content. Fluorescence polarization studies were carried out using 1,6-diphenyl-1,3, 5-hexatrien. Remarkable differences in the fatty acid pattern of the proximal and the distal colon were seen. Due to a higher content of oleic acid the saturation index of the apical membranes of the proximal colon is lower compared to that of the apical membranes of the distal colon (0.34 +/- 0.03 vs 0.42 +/- 0.05). The cholesterol content of the apical membranes of the proximal colon is markedly higher than that of the apical membranes of the distal colon (3.42 +/- 0.14 vs 1.88 +/- 0.29 mol/mol PL). There are no differences in the fluidity of these apical membranes. We assume a balancing mechanism between the cholesterol content and the amount of saturated PL-fatty acids.     

Localization of cholesterol in the colonic epithelium of the guinea pig: regional differences and functional implications

Summary It is generally accepted that variations in membrane cholesterol content affect the fluidity of the bilayer, thus altering its permeability. In the biological membranes, in physiological conditions, a high cholesterol content rigidifies the bilayer decreasing its permeability, a lower cholesterol content induces the opposite effect by increasing the permeability. Since differences in the epithelial permeability for short chain fatty acids have previously been demonstrated in the proximal and distal colon of the guinea pig, these two regions were investigated to establish whether differences in membrane cholesterol content of the absorbing cells can be demonstrated. Freeze-fracture replicas of filipin-treated colonic tissue were used. The results show that in the proximal colon the density of filipin cholesterol complexes located on the luminal plasma membrane of the columnar absorbing cells was significantly higher (about twice) than in the distal colon. Therefore the lower amount of cholesterol present in the membrane of the absorbing cells in the distal colon indicates a greater fluidity of the membranes of the epithelial cells in this region. Such fluidity could be correlated to the higher absorption rates of shortchain fatty acids characteristic of this region.     

Protein-lipid interactions in antipodal plasma membranes of rat colonocytes

The isolation of apical membranes from rat proximal colonic epithelial cells is described. Differential centrifugation yielded a ‘crude’ membrane fraction which was further purified using sucrose density centrifugation. The final membrane fraction was enriched 20–28-fold over homogenate in alkaline phosphatase and cysteine-sensitive alkaline phosphatase specific activities. Lipid-protein interactions and lipid dynamics examined in apical and basolateral membranes prepared from colonocytes demonstrated: (1) apical membrane, as assessed by steady-state fluorescence polarization studies have a low lipid fluidity; (2) colonic basolateral membranes possess a greater lipid fluidity than apical membranes; (3) compositional differences in these antipodal membranes appear to explain these differences in lipid fluidity; (4) fluorescence polarization studies using diphenylhexatriene detect a thermotropic transition at 21–23°C in apical membranes and liposomes prepared from lipid extracts of these membranes; (5) alkaline phosphatase and l-cysteine-sensitive alkaline phosphatase activities appear to be functionally dependent on the physical state of the apical membrane's lipid.     

Cytochemical demonstration of guanylate cyclase activity in cardiac muscle. Preferential localization at sarcolemma and junctional sarcoplasmic reticulum

The localization of guanylate cyclase activity was cytochemically studied in heart tissue from guinea pig and pigeon. The method, based on a lead precipitation technique with GPPNHP as the substrate, was tested by quantitative biochemical analysis. The data obtained showed that in heart homogenates GPPNHP is an acceptable substrate for guanylate cyclase. The guanylate cyclase activity of glutaraldehyde prefixed heart tissue was also measured in the presence of 2 mM lead nitrate, in 30% of the untreated control hearts. The residual guanylate cyclase responded to the addition of sodium nitroprusside with a 7-fold increase in its activity. Furthermore, the guanylate cyclase requirement for Mn2+ ions was so changed by this activator that Mg2+ was as active as Mn2+. In heart muscle cells of guinea pigs and pigeons the plasma membrane of the sarcolemma and the junctional sarcoplasmic reticulum are the precipitation sites of the reaction product. In guinea pig hearts the T-tubule membranes were likewise covered with precipitates. Sodium nitroprusside stimulation of guanylate cyclase activity was indicated by increased precipitation and by shortening of the incubation time.     

Identification of insulin receptors on the mucosal surface of colon epithelial cells

Brush-border membranes were isolated from the mucosal surface of rabbit proximal colon epithelial cells by a procedure involving Ca2+ precipitation. Ouabain-insensitive K+-phosphatase, a marker enzyme for the colon brush-border membrane, was enriched 17-fold by this technique, while no enrichment was observed in the activity of ouabain-sensitive K+-phosphatase, a marker for the basal-lateral membrane. Insulin binding studies revealed a dose-dependent inhibition of 125I-insulin binding with porcine insulin and approximately 4 X 10(-9) M insulin was required to produce 50% inhibition of 125I-insulin binding, while desoctapeptide insulin, insulin-like growth factor I, and A chain of insulin had less effect on 125I-insulin binding. This is the first demonstration of the existence of high-affinity insulin binding sites on the brush-border membrane of mammalian colon epithelial cells. Subsequent studies with the cross-linking agent disuccinimidyl suberate confirmed the presence of insulin binding sites in these membranes and autoradiography of polyacrylamide gels revealed that the binding subunit of the colon epithelial cell brush-border insulin receptor is similar in size to that observed in hepatic tissue. Interestingly, the insulin binding capacity/mg of protein of this preparation is high, suggesting that large numbers of insulin receptors are present in vivo on the mucosal surface of colon epithelial cells. The potential physiological role of these previously unrecognized insulin receptors is discussed.     

A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells

Murine mAbs were produced against purified microvillus membranes of rat colonocytes in order to establish a marker protein for this membrane. The majority of antibodies binding to the colonic microvillus membrane recognized a single protein with a mean apparent Mr of 120 kD in both proximal and distal colon samples. The antigen is membrane bound as probed by phase-partitioning studies using Triton X-114 and by the sodium carbonate extraction procedure and is extensively glycosylated as assessed by endoglycosidase F digestion. Localization studies in adult rats by light and electron microscopy revealed the microvillus membrane of surface colonocytes as the principal site of the immunoreaction. The antigen was not detectable in kidney or liver by immunoprecipitation but was present in the small intestine, where it was predominantly confined to the apical membrane of crypt cells and much less to the microvillus membrane of differentiated enterocytes. During fetal development, the antigen appears first in the colon at day 15 and 1-2 d later in the small intestine. In both segments, it initially covers the whole luminal surface but an adult-like localization pattern develops soon after birth. The antibodies were also used to develop a radiometric assay for the quantification of the antigen in subcellular fractions of colonocytes in order to assess the validity of a previously developed method for the purification of colonic brush-border membranes (Stieger, B., A. Marxer, and H.P. Hauri. 1986. J. Membr. Biol. 91:19-31.). The results suggest that we have identified a valuable marker glycoprotein for the colonic microvillus membrane, which in adult rats may also serve as a marker for early differentiation of enterocyte progenitor cells in small-intestinal crypt cells.     

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.     

Ontogeny of proximal colon basolateral membrane lipid composition and fluidity in the rabbit.

Basolateral membranes from rabbit proximal colon were prepared from isolated colonocytes throughout postnatal maturation, using a modification of published techniques. In suckling (14-20 day) and post-weaning/mature (35-49 day) animals, membranes were purified approx. 10-fold, based upon the enrichment of ouabain-sensitive, sodium-potassium dependent adenosine triphosphatase activity. Membrane lipid analyses demonstrated age-dependent increases in total cholesterol and the cholesterol/phospholipid molar ratio, as well as decreases in phosphatidylethanolamine content and the fatty acid unsaturation index. Fluidity of basolateral membranes and membrane liposomes, determined from fluorescence anisotropy measurements using the lipid probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid, demonstrated significant, ontogenic decreases in fluidity; and, additional studies showed that fluidity changes occurred early in the weaning period (by day 24 postnatally). Arrhenius plots of liposome anisotropies suggested a bilayer lipid thermotropic transition temperature of 22 degrees C in sucklings 26 degrees C in mature rabbits. These findings demonstrate that ontogeny of colonic basolateral membranes is associated with significant modulations in lipid composition and fluidity.     

Differentiation-dependent induction of CYP1A1 in cultured rat small intestinal epithelial cells,colonocytes, and human colon carcinoma cells: basement membrane-mediated apoptosis

Rat small intestinal epithelial cells and human colon adenocarcinoma cells cultured on Matrigel expressed the differentiation specific enzyme, sucrase-isomaltase, as determined by indirect immunofluorescence. Rat small intestinal epithelial cells, rat colonocytes, and human colon adenocarcinoma cells developed an altered morphology when cultured on Matrigel and became apoptotic within 24-48 h. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin caused a 2- and 5-fold induction, respectively, of ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity in rat small intestinal epithelial cells cultured on plastic was not detected. 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment caused a 14-fold induction of transfected, rat CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells cultured on Matrigel. Benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induced ethoxyresorufin-o-deethylase activity by 6- and 1.6-fold, respectively in rat colonocytes cultured on Matrigel. Induction of ethoxyresorufin-o-deethylase activity was not observed in rat colonocytes cultured on plastic. CYP1A1-promoter-luciferase activity was induced 3-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat colonocytes cultured on Matrigel. Induction of CYP1A1-promoter-luciferase activity in rat small intestinal epithelial cells or rat colonocytes cultured on plastic was not observed. Ethoxyresorufin-o-deethylase activity in human colon adenocarcinoma cells, cultured on either plastic or Matrigel, was induced 7-fold by benzo[a]pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced ethoxyresorufin-o-deethylase activity was 2-fold greater in human colon adenocarcinoma cells cultured on Matrigel compared to cells cultured on plastic. Extracellular matrix-mediated differentiation and apoptosis of intestinal cells provide in vitro systems for study of the regulation of CYP1A1 expression, carcinogen activation in the gut and mechanism(s) of apoptosis of colon cancer cells.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

Ultrastructural characterization of interstitial cells of Cajal associated with the submucosal plexus in the proximal colon of the guinea pig

Interstitial cells of Cajal (ICC) associated with the submucosal (submucous) plexus (ICC-SP) in the proximal colon of the guinea pig were studied by immunohistochemistry and electron microscopy. Whole-mount stretch preparations with c-Kit immunohistochemistry revealed that a number of ICC-SP constituted a dense cellular network around the submucosal plexus. Some of these ICC-SP were observed in the vicinity of the muscularis mucosae in sections immunostained for c-Kit and α-smooth muscle actin. Ultrastructural observation demonstrated, for the first time, that ICC-SP of the proximal colon of the guinea pig retained typical ultrastructural characteristics of ICC repeatedly reported in association with the tunica muscularis of the gastrointestinal tract: a basal lamina, caveolae, many mitochondria, abundant intermediate filaments and the formation of gap junctions with the same type of cells. The most remarkable ultrastructural finding was the presence of thick bundles composed of the processes of ICC-SP connected to each other via large gap junctions. These ICC-SP might be involved in the main mucosal functions of the proximal colon of the guinea pig, namely the transportation of water and electrolytes, possibly via their involvement in the spontaneous contractions of the muscularis mucosae.     

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.     

Glucose metabolism in proliferating epithelial cells from the rat colon.

1. The effects of fasting and fasting followed by refeeding on the activities of the oxidative pentose pathway (OPP) and the tricarboxylic acid cycle (TCA) in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]glucose and [6-14C]glucose, respectively. 2. Refeeding after a fast induced a 2-3-fold increase in glucose flux through the OPP and TCA cycle and the degree of change was similar in colonocytes from the proximal and distal colon. 3. Butyrate at a concentration of 40 mM inhibited the OPP by 20-30% (P less than 0.05) but had no effect on the activity of the TCA cycle. Glutamine at a concentration of 2 mM decreased the glucose flux through both the OPP and the TCA cycle by 30-50% (P less than 0.05). 4. Production of 14CO2 from the oxidation of butyrate or glucose indicated that the former was 5-7 times more active in colonocytes from fasted rats. After refeeding, however, butyrate utilization was similar to fasting values in the proximal colon but significantly lower (P less than 0.05) in the distal colon.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Atrial natriuretic factor activates membrane-bound guanylate cyclase of chief cells

The present study examined the effect of atrial natriuretic factor (ANF) on cGMP generation by dispersed chief cells from guinea pig stomach. ANF caused a rapid dose-dependent increase in cGMP, a 7-fold increase in cGMP caused by 1 microM ANF, with or without 3-isobutyl-1-methylxanthine present. Methylene blue reduced cGMP in response to nitroprusside but not ANF. Guanylate cyclase activity of a chief cell membrane fraction doubled in response to ANF, but was not affected by nitroprusside. ANF had no effect on guanylate cyclase activity of the soluble fraction of lysed chief cells. Dose-response curves for whole cell cGMP production and membrane guanylate cyclase activity in response to ANF were closely related. These data indicate that ANF increases chief cell cGMP production by activating particulate guanylate cyclase, providing functional evidence that chief cells possess surface membrane receptors for ANF.     

Isolation and characteristics of the plasma membrane fraction from the swine myometrium

An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP-dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+-accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Fucoidin inhibits attachment of guinea pig spermatozoa to the zona pellucida through binding to the inner acrosomal membrane and equatorial domains

Our previous study has shown that fucoidin, an algal heteropolysaccharide, is a potent inhibitor of sperm-zona binding in the guinea pig, hamster and human. To visualize the surface site of fucoidin binding, a biotinated derivative (B-Fuc) of the native fucoidin was prepared. B-Fuc retained the inhibitory activity and was used in conjunction with FITC-avidin to localize its binding sites on guinea pig spermatozoa using fluorescence microscopy. In living acrosome-reacted spermatozoa, B-Fuc bound predominantly to the inner acrosomal membrane and equatorial segment domains. The binding was effectively competed by a 10-fold excess of native fucoidin, but not by a 10-fold excess of heparin or a 20-fold excess of biotinated normal rabbit serum IgG. B-Fuc binding patterns on dead spermatozoa were quite different from that of living spermatozoa. The post-acrosomal region, rather than the inner acrosomal membrane and equatorial domains, was intensely labeled. This indicates the importance of using living cells in assessing true surface binding sites whenever possible. We conclude that the inner acrosomal membrane and/or equatorial domains are critical for zona binding in the guinea pig.