Food Hydrocolloids 25 (2011) 1618e1626
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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
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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
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(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
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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.
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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
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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).
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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. We thank Rita Wallén (Dept. of Biology,
Lund University) for taking the electron micrographs, Christer
Larsson and Adine Karlsson (Dept. of Biochemistry and Structural
Biology, Lund University) for spinach plasma membranes.
1625
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