Food Hydrocolloids 25 (2011) 1618e1626 Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd Pigments protect the light harvesting proteins of chloroplast thylakoid membranes against digestion by gastrointestinal proteases Sinan C. Emek a, Hans -Erik Åkerlund a, Maria Clausén a, Lena Ohlsson b, Björn Weström c, Charlotte Erlanson-Albertsson d, Per-Åke Albertsson a, * a Department of Biochemistry and Structural Biology, Chemical Centre, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Department of Clinical Science, Medicine (Gastroenterology and Nutrition), Lund University, SE-221 85 Lund, Sweden Department of Biology, Lund University, SE-223 62 Lund, Sweden d Department of Experimental Medical Science, Appetite Control Unit, BMC, Lund University, SE-221 84 Lund, Sweden b c a r t i c l e i n f o a b s t r a c t Article history: Received 25 August 2010 Accepted 13 December 2010 Chloroplast thylakoid membranes inhibit pancreatic lipase/colipase activity in vitro and, when included in food, induce satiety signals. As thylakoid membranes themselves are nutrients, containing lipids and proteins, it is of interest to study the digestion of thylakoids by enzymes of the gastrointestinal tract. Thylakoid membranes were treated with pepsin, trypsin, gastric and pancreatic juice at 37 C and the resulting enzymatic breakdown was analyzed by gel electrophoresis, electron microscopy and mass spectroscopy. In all cases, several of the proteins were degraded within half an hour, while the main parts of the pigmenteprotein complexes were resistant for hours. Oil emulsified thylakoids were more resistant towards the enzymatic breakdown. Electron microscopy demonstrated that, after treatments, the thylakoids still remained in a membrane vesicular form. The capacity of thylakoid membranes to inhibit the lipase/colipase activity was partly reduced in all cases. About 50% of the inhibition capacity remained after treatment with pancreatic juice when the thylakoids were present in an oil emulsion. Delipidated thylakoids and plasma membranes, which lack the photosynthetic pigments, were degraded rapidly by pancreatic juice. Conclusion: The pigments, closely bound to the trans-membrane helices of thylakoid membrane proteins protect these from digestion by pepsin, trypsin, gastric and pancreatic juice. This supports the notion that a substantial inhibition of lipase/colipase takes place during the first 2 h in the intestine resulting in a retardation and prolongation of lipolysis in vivo. Ó 2010 Elsevier Ltd. All rights reserved. Keywords: Thylakoids Lipase Colipase Pepsin Trypsin Emulsion Gastric and pancreatic juice 1. Introduction The chloroplast thylakoid membranes account for a large part of the cell membranes of green leaves. They are responsible for conversion of light energy into ATP and NADPH which are used in the assimilation of carbon dioxide for the production of carbohydrate. Thylakoids are the most abundant biological membranes on earth. The thylakoid membrane forms a physically continuous threedimensional network of paired membranes enclosing between them the lumen and separating it from the surrounding stroma of the chloroplast (Albertsson, 2001; Decker & Boekema, 2005; Eberhardt, Abbreviations: LHC I or II, light harvesting chlorophyll a/b protein complex I or II; PS I, photosystem I; PS II, photosystem II; CCK, cholecystokinin; NaTDC, sodium taurodeoxycholate; DMSO, dimethyl sulfoxide; MALDIeTOF, matrix assisted laser desorption/ionizationetime of flight; PMSF, phenylmethylsulfonyl fluoride; TFA, trifluoroacetic acid. * Corresponding author. Tel.: þ46 46 2228190; fax: þ46 46 2224116. E-mail address: per-ake.albertsson@biochemistry.lu.se (P.-Å. Albertsson). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.12.004 Finazzi, & Wollman, 2008; Nelson & Ben-Sham, 2004). The membrane contains more than a hundred different proteins involved in the photosynthetic electron transport. The membrane proteins are both intrinsic i.e. membrane spanning and extrinsic i.e. attached to the surface of the membrane. The main intrinsic membrane protein complexes are PS I, LHC I, PS II, LHC II, cytochrome b/f, and ATP synthase. Together with their bound pigments, chlorophyll and carotenoids (Juhler, Andreasson, Yu, & Albertsson, 1993; Schmid, 2008) the membrane proteins account for approximately 70% of the thylakoid mass. The membrane lipids, galactolipids, phospholipids, sulfolipids (Duchene & Siegenthaler, 2000) plastoquinones, tocopherols and phylloquinones (Munné-Bosch & Alegre, 2002; Zbierzak et al., 2009) account for the remaining 30% of the thylakoid dry mass. Plastoglobules, attached to the thylakoid membrane, also contain proteins and lipids (Bréhélin, Kessler & vanWijk, 2007). In addition to the asymmetry across the membrane, there is a lateral separation of different regions with different biochemical composition and function (Albertsson, 2001; Bukhov & Carpentier, S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 2004; Danielsson & Albertsson, 2009; Danielsson, Albertsson, Mamedov, & Styring, 2004). Stacked regions, called grana, are enriched with photosystem II (PSII), while photosystem I (PSI) and ATP synthase are localized in the stroma exposed regions: stroma lamellae, end membranes and grana margins. The cytochrome bf complex is found all over the membrane (Albertsson, Andreasson, Svensson, & Yu, 1991). A substantial fraction of the membrane lipids are strongly bound to the intrinsic protein complexes and form a solvation shell around the membrane spanning part of them (Minoda et al., 2002; Páli, Garab, Horváth, & Kóta, 2003). Thylakoids have the capacity to inhibit the activity of pancreatic lipase, the main enzyme acting together with colipase during lipolysis of fat in the intestine (Albertsson et al., 2007). Lipase and colipase form a 1:1 complex the three-dimensional structure of which has been determined (Whitcomb & Lowe, 2007 and references therein). Lipase alone is easily inhibited by proteins and detergents, but together with colipase and bile salts the activity of lipase under physiological conditions is retained (Borgström & Erlanson-Albertsson, 1982; Erlanson-Albertsson, 1992). It is mainly the protein fraction of the thylakoids which have the capacity to inhibit lipaseecolipase based on the fact that delipidated thylakoids inhibit lipaseecolipase to about the same extent as intact thylakoids (Albertsson et al., 2007). The mechanism behind the inhibition is due to complex interactions between the three components, lipid droplets, the lipaseecolipase complex and the thylakoids: 1) The lipaseecolipase complex has a strong affinity for binding to its substrate, the lipid droplets, 2) the thylakoids have strong affinity to the surface of the lipid droplets and 3) the lipaseecolipase has a strong affinity for the thylakoid membrane (Albertsson et al., 2007). This “Trilogy” of interaction means that the lipaseecolipase complexes are sterically hindered to reach their substrate by the thylakoids bound to the lipid droplets at the same time as the lipaseecolipase complexes are bound to the thylakoids. When included in food, thylakoids induce satiety hormones such as cholecystokinin (CCK) leptin and enterostatin while reducing the hunger peptide ghrelin concomitant with reduced serum triglyceride and body fat. This has been demonstrated in long term studies on mice (Köhnke, Lindbo et al., 2009; Köhnke, Lindqvist et al., 2009) and rats (Albertsson, et al., 2007; Emek et al., 2010) and short term studies on humans (Köhnke, Lindbo et al., 2009; Köhnke, Lindqvist et al., 2009). These in vivo results are interpreted as due to a prolongation of the lipid digestion inducing satiety (Beglinger & Degen, 2004; Ritter, 2004). Since thylakoids are composed of proteins and lipids the question then arises how rapidly they are broken down by gastrointestinal enzymes. In this work e by simulating the gastrointestinal digestion process e we have studied the degradation of thylakoids and their effect on lipase/colipase activity. 2. Materials and methods 2.1. Preparation of thylakoid membranes Thylakoid membranes were prepared from spinach (Spinacia oleracea) leaves as described (Andreasson, Svensson, Weibull, & Albertsson, 1988; Emek et al., 2010). Protein was determined by BIO-RAD DC protein assay kit and chlorophyll according to (Porra,Thompson, & Kriedemann, 1989). 2.2. Delipidation of thylakoid membranes Purified thylakoid membranes (107 ml of 3.9 mg/ml chlorophyll) were mixed with 428 ml of ice-cold acetone by intensive magnetic stirring for 1 min followed by mild mixing for 5 min. The mixed 1619 solution was allowed to settle for 10 min 50% of supernatant was withdrawn and replaced with the same volume of ice-cold acetone during magnetic stirring. The solution was allowed to settle for another 10 min and then 50% of supernatant was withdrawn. The rest of the solution was centrifuged for 10 min at 5000 rpm. The supernatant was discarded and the pellet resuspended in 415 ml of 50 mM phosphate buffer, pH 7.1, carefully homogenized with a glass potter and allowed to stand for 20 min. The sample was then centrifuged for 10 min at 7500 rpm. The pellet now contained delipidated insoluble thylakoid membrane proteins. 2.3. Sodium dodecyl sulfateepolyacrylamide gel electrophoresis (SDSePAGE) Samples for gel electrophoresis analysis were diluted 1:4 with NuPAGE-LDS sample buffer. For each well, the same amount of protein (30 mg) was loaded. PageRulerÔ Prestained Protein Ladder (10 ml) from Fermentas was used as a protein standard. NuPAGE Novex 4e12% gradient midi pre-cast gels were used to carry out the SDSePAGE with NuPAGE e MES 2-(N-morpholino) ethane sulfonic acid e SDS as a running buffer. The conditions of electrophoresis were 200 V for 55 min. The gel was stained in coomassie brilliant blue R-250. 2.4. Mass spectrometry Mass spectrometry analysis was carried out as described (Emek et al., 2010; Everberg, Peterson, Rak, Tjerneld, & Emanuelsson, 2006). 2.5. Pancreatic lipase/colipase activity Porcine pancreas lipase, type VI-S, and porcine pancreas colipase were from Sigma. The lipase/colipase activity was determined by pH stat titration apparatus (TIM854 model Radiometer Analytical SAS, Cedex France). Tributyrine was used as substrate and 0.1 M NaOH for titration. 15 ml of assay buffer, containing 2 mM Tris-maleate (pH 7), 0.15 M NaCl, 1 Mm CaCl2 and 4 mM sodiumtaurodeoxicholate (NaTDC), was mixed with 0.5 ml tributyrine as described (Erlanson-Albertsson, Larsson, & Duan, 1987). Then, 10 ml of lipase solution, 1 mg/ml in assay buffer (see above) and the same amount of colipase in aqueous solution were added. Consumption of NaOH (mmol/min) was taken as activity of lipase/colipase. The tributyrine was omitted in the assay mixture when the lipase/ colipase activity was measured on thylakoid emulsions since tributyrine was already present in the emulsion. 2.6. Treatment with proteases and pancreatic juice 2.6.1. Thylakoids alone Pepsin, porcine gastric mucosa, lyophilized powder, and trypsin, type XI from bovine pancreas, lyophilized powder, were obtained from SigmaeAldrich, Pure porcine pancreatic juice was collected from anesthetized pancreatic duct-cannulated pigs (10e20 kg b wt), during basal conditions and during stimulation with secretin and CCK, pooled and stored frozen at 20 C until used (Rengman, Weström, Ahrén, & Pierzynowski, 2009). Human gastric juice, a gift from Dr Berit Sternby, BMC, Lund University, and human pancreatic juice, a gift from Dr Jan Axelsson, Dept of Surgery at University Hospital of Malmö, was collected from a drainage tube in the pancreatic duct due to a cyst. Purified thylakoid membranes (0.33 ml), containing 3 mg/ml chlorophyll, were mixed with 0.17 ml of various amounts (see figure texts) of pepsin, trypsin, porcine pancreatic juice, human gastric and pancreatic juice. In the case of pepsin and human gastric juice treatments, 0.5 ml of water was used and the pH was adjusted to 2.0 with HCl. For trypsin or pancreatic juice, 0.5 ml of buffer 1620 S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 (4 mM Tris-maleate pH 7.0, 8 mM NaTDC, 2 mM CaCl2 and 0.3 M NaCl) were used. All mixtures were incubated at 37 C. Trypsin and pancreatic juice proteases were inactivated with 1 mM PMSF. Pepsin and human gastric juice were inactivated by adjusting pH to 7.0. 2.6.2. Thylakoids in oil emulsion 2.6.2.1. Pepsin. 0.33 ml of varying amount of thylakoid membranes, 0.17 ml pepsin, 0.5 ml tributyrine and 0.5 ml water were mixed and pH was adjusted to 2.0 with HCl. The mixture was homogenized by using HeidolphÒ SilentCrusher S homogenizer. Emulsions were incubated at 37 C for 1 h. Pepsin was inactivated by adjustment to pH 7.0. 2.6.2.2. Trypsin and pancreatic juice. 0.33 ml of varying amount of thylakoid membranes, 0.17 ml trypsin or pancreatic juice, 0.5 ml tributyrine and 0.5 ml buffer (6 mM Tris-maleate pH 7.0, 12 mM NaTDC, 3 mM CaCl2 and 0.45 M NaCl]) were homogenized as described above for pepsin. Emulsions were incubated at 37 C for 2 h. 1 mM PMSF was used for inactivation of proteases. 2.7. Electron microscopy (EM) Samples for EM were mainly prepared as described above with some modifications. Samples with emulsions were prepared with rapeseed oil instead of tributyrine. All samples were fixed first with 2.5% (w/v) glutaraldehyde in 0.15 M cacodylate buffer then imbedded in Epon and finally stained in 3% (v/v) uranyl acetate and lead citrate. 2.8. Plasma membranes Plasma membranes from spinach (S. oleracea) leaves prepared as described (Larsson, Sommarin, & Widell, 1994) were a gift of Adine Karlsson, Dept. of Biochemistry and Structural Biology, Lund University. 3. Results 3.1. Treatments of thylakoid membranes After treatments with different proteases the thylakoids were analyzed by SDSePAGE. Thylakoid membrane proteins have been extensively characterized by SDSePAGE and the molecular weight of the monomers of the different intrinsic membrane protein complexes is well known (Barros & Kuhlbrandt, 2009; Liu et al., 2009; Nelson & Ben-Sham, 2004; Schmid, 2008) and also the location of the monomers in the SDSePAGE gels (Andreasson et al., 1988; Emek et al., 2010). Isolated LHC, the major pigment complex, shows two bands around 25e27 kD in the gel (Andersson & Albertsson, 1981). In addition the capacity of the treated thylakoids to inhibit lipase/colipase activity was determined. 3.1.1. Pepsin treatment The effect of pepsin on the thylakoid membrane proteins as visualized by gel electrophoresis is shown in Fig. 1A. Most of the weakly stained proteins were degraded after 60 min at 37 C by 0.5 mg/ml of pepsin. Two bands stand out as more resistant towards the pepsin treatment. One is a broad band representing the light harvesting proteins (LHC I and II) around 25 kDa and the other just below 55 kDa (not identified). Except for a slight reduction in molecular weight, these two bands appeared to withstand pepsin treatment for at least 1 h. The EM-picture of pepsin treated thylakoid membranes in oilewater emulsion (Fig. 1B) shows that thylakoids were in the form of both stacked grana-like structures and swollen membrane vesicles, attached to the oil surface, much like untreated thylakoids attached to oil droplets see Figure 3A in Albertsson et al. (2007). 3.1.2. Trypsin treatment Gel electrophoresis on the trypsin treated thylakoids (Fig. 2A) show that several of the proteins were degraded after 2 h treatment. However, the LHC 1 and II proteins (25 kDa band) and the 55 kDa bands were essentially resistant except for a slight reduction of molecular weight and a split of the 25 kDa band into two bands. The EM-picture of trypsin treated thylakoids in oil emulsion (Fig. 2B) shows that the thylakoids remained in a somewhat swollen membrane-vesicle form attached to the oil surface. 3.1.3. Pancreatic juice treatment Pancreatic juice contains a large number of proteases, lipases, nuclease and amylase. Their effects on thylakoids are shown in Fig. 3A. After 2 h, most of the proteins were degraded but the LHC I and II and the 55 kDa bands were still visible. There was, however, a larger down-shift of LHC to a lower molecular weight in the case of pancreatic juice compared to the pepsin or trypsin treatments (Figs. 1A and 2A) indicating that a larger part of the LHC proteins had been split off. Remaining polypeptide bands of pancreatic juice treated thylakoids (Fig. 3A) were identified with MALDIeTOF mass spectrometry (spectra not shown). The proteins most resistant towards the pancreatic juice were found to be the pigmenteprotein complexes, PSI and PSII with their respective light harvesting complexes LHC I and LHC II, but also the alpha and beta subunits of Fig. 1. A) SDSePAGE of thylakoid membranes treated with pepsin. Thylakoid membranes (1 mg/ml chlorophyll) were treated with varying amount of pepsin at 37 C for 1 h at pH 2.0. B) EM-picture of thylakoid membranes (1 mg/ml chlorophyll) treated with pepsin (1 mg/ml) at 37 C for 1 h in an oilewater emulsion, pH 2.0. The thylakoid membranes are attached to the oil surface. S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 1621 Fig. 2. A) SDSePAGE of thylakoid membranes (1 mg/ml chlorophyll) treated with 300 mg trypsin for different times at 37 C. B) EM-picture of thylakoid membranes (1 mg/ml chlorophyll) treated with trypsin (1 mg/ml) at 37 C for 2 h in oilewater emulsion with 4 mM NaTDC. The thylakoid membranes are unfolded compared to the ones in Fig. 1B. ATP synthase. Treatment with human pancreatic juice gave almost identical results (not shown). The EM-picture of the porcine pancreatic juice treated thylakoids (Fig. 3B) shows that the vesicles were more unfolded and irregular compared to the vesicles after pepsin or trypsin treatment (Figs. 1B and 2B). This is probably due to the presence of several enzymes and bile salts. The effect of the bile salts without proteases or other enzymes on the thylakoid membranes are shown in Fig. 4. 3.1.4. Gastric juice treatment followed by pancreatic juice treatment To simulate the in vivo digestion process we treated the thylakoids with human gastric juice for 1 h at pH 2.0 followed by treatment with human pancreatic juice for 2 h at pH 7.0 (Fig. 5). The bands, just below 25 kDa, representing the LHC I and II proteins are still present pointing to a strong protection of the pigments towards digestion of these proteins. Identical results were obtained with porcine gastric and pancreatic juices. 3.2. Pancreatic juice treatment of plasma membranes as an example of non-pigmented membranes The protein degradation of plasma membranes, treated by porcine pancreatic juice, was very rapid. Already after 5 min treatment almost all proteins were degraded (Fig. 6). 3.3. The effect on the capacity of thylakoids to inhibit lipase/colipase It has earlier been shown that thylakoids inhibit the pancreatic lipase/colipase activity (Albertsson, 2001). Fig. 7 shows a typical inhibition curve. The lipase/colipase activity was reduced with increasing amount of thylakoids, down to a plateau of about 20% of the activity in the absence of thylakoids i.e. the inhibition capacity of the thylakoids is 80% (Fig. 7). 3.3.1. Pepsin treatment Treatment of thylakoids with pepsin reduced their capacity to inhibit the lipase/colipase activity in a dose dependent way (Fig. 8A). The lipase/colipase activity increased from 20% up to a plateau value of about 50% of the lipase/colipase activity in the absence of thylakoids (100%) i.e. the inhibition capacity of the thylakoids was reduced from 80% to about 50%. When thylakoids were included in an emulsion during the pepsin treatment the lipase/colipase activity reached a value of 30% (Fig. 8A). This means that the inhibition capacity of the pepsin treated thylakoids in oil emulsion was 70% of the activity in the absence of thylakoids. Thus, the results show that the oil emulsion had a protective effect on the thylakoids against pepsin. Fig. 3. A) SDSePAGE picture of thylakoid membranes treated with porcine pancreatic juice (0.5 mg/ml) at 37 C for different times. Down pointing arrows on the gel picture show proteins identified with MALDIeTOF ms/ms analysis: 1) Photosystem I P700, 2) Pancreatic alpha-amylase, 3) ATP synthase subunit alpha, Photosystem I, P700, 4) LHC Proteins, 5) Pancreatic alpha-amylase. Note the breakdown of the proteins to the polypeptide size of about 2 kDa. B) EM-picture of thylakoid membranes (1 mg/ml chlorophyll) treated with pancreatic juice (0.5 mg/ml) at 37 C for 2 h in an oilewater emulsion with 4 mM NaTDC. The thylakoid membranes attached to the oil surface are unfolded and swollen. The dark bodies represent plastoglobules. 1622 S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 Fig. 6. SDSePAGE of spinach plasma membranes (5 mg/ml protein) treated with porcine pancreatic juice (0.5 mg/ml) for different times at 37 C. Plasma membranes, lacking photosynthetic pigments, are rapidly degraded. Fig. 4. EM-picture of thylakoid membranes incubated in 4 mM NaTDC at 37 C for 2 h. The dark bodies represent plastoglobules. 3.3.2. Trypsin treatment Trypsin treatment also reduced the capacity of thylakoids to inhibit lipase/colipase, much in the same way as pepsin (Fig. 8B). Already at 0.5 mg/ml of trypsin treatment the lipase/colipase activity reached a value of about 45%. The oil emulsion protected the thylakoids so they kept an inhibition capacity of 70% (Fig. 8B). chain of ATP synthase (Fig. 9A). These results demonstrate that the lipids and/or pigments protect the pigment containing protein complexes of PS I and II complexes and LHC I and II. 3.4.2. The effect on the capacity of delipidated thylakoids to inhibit lipase/colipase This is shown in Fig. 10. After 2 h treatment, the delipidated thylakoids in oil emulsion had approximately 80% of the inhibition capacity on the activity of lipase/colipase while just above 70% without emulsions. 4. Discussion 3.3.3. Gastric and pancreatic juice treatment Human gastric juice followed by human pancreatic juice reduced the capacity of the thylakoids to inhibit lipase/colipase activity down to 35% (Fig. 8C). In an oil emulsion about 50% of the inhibition capacity still remained even after 2 h treatment at 37 C. Identical result was obtained after treatment with porcine gastric juice followed by porcine pancreatic juice (not shown). 3.4. Treatments of delipidated thylakoids 3.4.1. Gel electrophoresis The effect of treatment with pepsin, trypsin or pancreatic juice on delipidated thylakoids showed that, in each case, after 1 h of treatment, almost all proteins were degraded (Fig. 9A, B and C) except for a polypeptide band around 50 kDa identified as the alpha Fig. 5. SDSePAGE of thylakoids treated first with human gastric juice (0,25e1.0 mg/ ml) 1 h, 37 C, pH 2.0, then human pancreatic juice (0,25e1.0 mg/ml) 2 h, 37 C, pH 7.0. The dominating proteins of thylakoid membranes are the proteins associated with the two photosystems PSI and PSII which together with their light harvesting complexes, LHC I and LHC II, account for more than 80% of the thylakoid protein mass. As shown in Figs. 1, 2 and 3 and 5 these proteins are relatively resistant towards degradation by proteases while the other, the non-pigmented proteins, except the alpha and beta subunits, are degraded rapidly by pepsin, trypsin or pancreatic juice. The plasma membranes which lack the pigments are degraded almost instantaneously. 4.1. “Shaving” of the thylakoids To explain the different effects of proteases, gastric and pancreatic juice on the pigment containing membrane proteins one has to Fig. 7. Inhibition curve of thylakoid membranes on the activity of lipase/colipase. Thylakoids having 1 mg chlorophyll reduces the lipase/colipase activity down to 20% of the activity without thylakoids (100%) i.e. they have an inhibition capacity of 80%. The same amount of added thylakoids (1 mg chlorophyll) was used as starting point in the experiments of Fig. 8. Figure is redrawn from (Albertsson et al., 2007). S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 1623 Fig. 8. Effect of pepsin, trypsin and gastric/pancreatic juice treatment (37 C) of thylakoid membranes (with and without emulsions) on the activity of lipase/colipase. The incubation times were 1 h for treatment of pepsin and 2 h for treatment of trypsin and pancreatic juice. Fig. 9. SDSePAGE pictures of delipidated thylakoids treated (37 C) with A) pepsin (1 mg/ml), B) trypsin (0.3 mg/ml) and C) pancreatic juice (0.5 mg/ml). 1624 S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 occur only on the outside stromal side since the loops on the inside luminal side are not available for the protease attack provided the thylakoid membrane is intact (Åkerlund & Jansson, 1981). Treatment with pancreatic juice alone (Fig. 3A) resulted in a faster migrating LHC monomer band compared to treatment with gastric juice followed by pancreatic juice (Fig. 5). The reason for this is not known. However, possible explanations could be a) pepsin cuts off the cleavage site for trypsin or other pancreatic juice proteases, b) the low pH may alter the structure of the LHC proteins causing aggregation of the exposed loops so that they are hidden for the pancreatic juice proteases attack. 4.2. Effect of pigments Fig. 10. The effect of delipidated thylakoid membranes treated (37 C) with pancreatic juice, with and without emulsions, on the activity of the L/CL. consider the three-dimensional structure of these different proteins (Amunts, Drory, & Nelson, 2007; Barber, 2002; Barros & Kuhlbrandt, 2009; Liu et al., 2009; Nelson & Ben-Sham, 2004). Both photosystems, PS I and PS II, with their respective light harvesting complexes, LHC I and LHC II, are intrinsic membrane proteins which dominate the thylakoid mass. The three-dimensional structure of the monomers of LHC I and LHC II has been determined with high resolution. Each monomer (25 kDa band) consists of one polypeptide chain with four membrane embedded helices (Fig. 11). Most probable, the proteases acted first on the N-terminal, external polypeptide chain on the stromal side of the thylakoid membrane vesicles. This polypeptide chain is 54 amino acids long, with several theoretical cleavage sites, and partial degradation of this fragment can explain the very slight reduction in the molecular weight of the 25 kDa bands observed in Figs. 1A and 2A. This membrane “shaving” will PSI and PSII and their light harvesting proteins contain several pigments, chlorophyll a and b and carotenoids, which are attached to the hydrophobic, membrane spanning helices (Schmid, 2008). In the case of the monomer of LHC II (Lhcb1) 14 chlorophylls and 4 carotenoids are interacting with the four membrane spanning, hydrophobic helices (Barros & Kuhlbrandt, 2009; Liu et al., 2009). The number of amino acids of these four helices is 102 i.e. a molecular mass of about 11 kDa. The 14 chlorophylls together with the 4 carotenoids have a molecular mass of about 15.8 kDa i.e. the mass of bound pigments exceeds that of the membrane spanning helices. In addition, some of the membrane lipids bind strongly to the protein complexes. Monogalactolipids, sulfolipids and phosphatidylglycerol are tightly bound to PSII (Minoda et al., 2002; Páli et al., 2003). Taken together, this mass of pigments and lipids around the membrane helices provide a barrier towards proteases to act on their substrate and thereby retard the digestion of the thylakoids. In contrast, plasma membranes which lack photosynthetic pigments and delipidated thylakoids, lacking most of the pigments, are degraded much faster compared to intact thylakoids. 4.3. Effect of fatty acids and bile salts Fatty acids are produced in vivo both in the stomach and the small intestine as a result of lipolysis. The fatty acids are incorporated into the thylakoid membrane. As a result the surface area of the thylakoids increases together with unstacking of the thylakoids (Shaw, Anderson, & McCarty, 1976). This is probably the reason why the thylakoids are much more swollen after treatment with pancreatic juice (Fig. 3B) compared to treatments with pepsin or trypsin (Figs. 1B and 2B). Bile salts, being amphiphilic, are probably also, like the fatty acids, incorporated into the thylakoid membrane and cause unfolding as shown in Fig. 4. This incorporation of fatty acids and bile salts may also contribute to the protection of thylakoids against proteases. Further issues to be investigated are whether membrane lipids protect membrane proteins against proteases and proteins protect lipids against lipases. 4.4. Effect on the capacity to inhibit lipase/colipase Fig. 11. Schematic representation of LHC II monomer (Lhcb1) embedded in the thylakoid membrane. The stroma side is on the outside and lumen side on the inside of the thylakoid membrane vesicles. Only the N-terminus external loop (54 amino acids) is easily available for proteolysis. The polypeptide chain is 267 amino acids long with 4 hydrophobic helices embedded in the membrane. The molecular mass of these membrane spanning helices is about 11 kDa Since14 chlorophyll and 4 carotenoids are attached to the helices (Barros & Kuhlbrandt, 2009; Liu et al., 2009; Schmid, 2008) the mass of these exceeds that of the helices. Together with some membrane lipids bound to the helices the pigments provide a barrier towards proteases to come in contact with their substrate. See (Barros & Kuhlbrandt, 2009; Liu et al., 2009) for a detailed structure of LHC. The capacity to inhibit lipase activity in vitro was reduced to about 50% in the case of treatment with pepsin or trypsin and to about 40% in the case of treatment with pancreatic juice. This could be due to the removal of some hydrophobic groups on the surface of the treated thylakoids leading to a reduced ability for the thylakoids to adsorb onto the lipid droplets and/or to less adsorption of lipaseecolipase onto the thylakoids. Alternatively the folding of the thylakoid membranes might be altered such that the exposed surface is reduced. The presence of oil in the form of emulsion protects the thylakoid membrane against degradation by the protease treatment more than in the absence of emulsion as demonstrated by SDSePAGE (not S.C. Emek et al. / Food Hydrocolloids 25 (2011) 1618e1626 shown). More importantly, the inhibition capacity is less reduced; only to about 70% in the case of pepsin or trypsin treatment and 50% in the case of treatment with gastric juice followed by pancreatic juice, Fig. 8. This can be explained in the following way: when the thylakoids are adsorbed onto the lipid droplets part of the thylakoid membrane surface will be less susceptible to pepsin, trypsin and other pancreatic enzymes; hence digestion will be slowed down. 4.5. General comments This study involves in vitro experiments. In the intestinal tract the situation is extremely complicated due to the large number of hydrolytic enzymes, at varying concentrations, and a large number of food components which requires completely different techniques for a relevant in vivo study. However, the results presented here show that the thylakoids due to their tight binding of pigments to the main membrane spanning proteins are remarkably resistant, even at 37 C, towards treatment with pepsin, trypsin, gastric and pancreatic juice and particularly so in the presence of an oil in water emulsion. The concerted action of gastric and pancreatic juice is expected to be most effective in breaking down the thylakoids. Pancreatic juice contains a whole battery of digestive enzymes such as trypsin, chymotyrpsin, elastase, lipase/colipase, carboxyl ester lipase, pancreatic lipase related proteins (PLRP 1 and 2), phospholipase A2 and alpha-amylase. Of these, carboxyl ester lipase and PLRP 2 are particularly interesting since they have broad substrate specificity (Whitcomb & Lowe, 2007). Both can hydrolyze galactolipids the main membrane lipids of the thylakoids (Andersson et al., 1994, 1996). It has been reported that PLRP 2 is not found in pig pancreas in contrast to human pancreas (de Caro et al., 2008). If so, since we found the same digestion pattern with human and pig pancreas juice, our results suggest that either the carboxyl ester lipase is the main enzyme hydrolyzing the galactolipids in pigs or that hydrolysis of galactolipids does not influence the digestion of thylakoids by proteases. 4.6. Conclusion Our results show that the pigment containing protein complexes of thylakoids are remarkably resistant towards breakdown by pepsin, trypsin and pancreatic juice. In addition a large part of the inhibition capacity of the thylakoids remains after the enzyme treatments. This suggests that a substantial inhibition of lipase/colipase can take place during the first 2 h in the intestine resulting in a retardation and prolongation of lipolysis in vivo. This in turn induces an increase of the satiety hormones CCK, leptin, enterostatin, and reduction of the hunger hormone grehlin as demonstrated in previous work (Albertsson et al., 2007; Emek et al., 2010; Köhnke, Lindbo et al., 2009; Köhnke, Lindqvist et al., 2009). After 2 h, however, the pigment containing protein complexes will be degraded and the dietary lipids will eventually be taken up by the small intestine. The net result is not a lasting inhibition but only a retardation of lipolysis resulting in an increase of satiety lasting over a longer time. Acknowledgements This work was funded by the Swedish Research Council, Royal Physiographic Society in Lund, Carl Trygger Foundation and Sven and Lilly Lawski Foundation. 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