Effect of β-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk

Biochimica ef Biophysics Acta, 1123 (19291) 151-155 0 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.00

151

BBALIP 53790

Effect of ~-la~toglobulin on the activi~ of pr~gastri~ lipase. A possible role for this protein in ruminant milk M.Dolores

Perez, Lourdes Sanchez, Paloma Aranda, Jose Manuel Ena, Rosa Oria and Miguel Calvo

Tecnologia y Bioqui’mica de 10s Alimentos, Facultad de Veterinaria, Unirersidad de Zaragoza, Zaragoza (Spain)

(Received 13 June 1991)

Key words: ~-Lactogiobulin; Pregastric lipase; Fatty acid; (Ruminants milk)

The interaction of bovine /3-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine /3-lactoglobulin displays only one high afftnity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 X lo6 and 2.3 X lo6 M -I, respectively. IIowever, other binding sites with low affhrity are also present. The existence of one high afIinity binding site is in accordance with the amount of fatty acids naturally bound to j3-lactoglobulin isolated from milk. The effect of fi-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of fi-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that j?-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.

Introduction /3-lactoglobulin is the major protein in the whey fraction of ruminant milk [l]. Its ~ncentration varies throughout lactation appearing at higher levels during the colostral period, about lo-20 mg/ml[2]. Although it had been thought that this protein occurs only in ruminant milks, it has been also detected in milk from non ruminant species as horse, pig [l], dog 131,kangaroo [4] and some aquatic mammals such a the dolphin [3] and whale [5]. However, milks from primates, rodents and lagomorphs appear to be devoid of /3lactoglobulin [3,6]. With the discovery in 1966 that cu-lactalbumin was a constituent of the lactose-synthetase [7] /%lactoglobulin remains the only major whey protein for which a biological role is still lacking. An interesting physico-chemical property of this protein is its ability to bind ‘in vitro’ some hydrophobic substances such as fatty acids [8,9] and retinol [lO,ll].

Correspondence: M. Calvo, Technologia y Bioquimica de 10s Alimentos, Facultad de Veterinaria, Un~ersidad de Zaragoza, Miguel Servet, 177,50013-Zaragoza, Spain.

Furthermore, ruminant ~-la~toglobuiins, isolated by non denaturing methods, have lipids physiologically bound, mainly long chain fatty acids [9,12]. The existence of preduodenal or prepancreatic lipases has been reported in various species including ruminants, rodents and lagomorphs 113,141. These enzymes are located either in the tongue, the pharyngeal area or the stomach depending on the species 113,141. In ruminants, this lipase cahed pregastric lipase, lingual lipase or triacylglycerol acyl hydrolase (EC 3.1.1.3) is present in the pharynx-glosso-epiglotic area and in the root of the tongue [14,15]. Its activity is higher in young animals and declines sharply when the animal becomes older [14,161. The preduodenal lipases participate in the fat digestion in the stomach [14]. Their optimun pH ranges from 4.0 to 6.0 and they have remarkable stability to low pH and pepsin degradation 117-191. The biological relevance of the preduodenal lipase is important to very young animals because their levels of pancreatic lipase and bile salts are low at this age [20,22]. Furthermore, among the digestive tract lipases, preduodenal lipases are unique in their ability to attack native milk fat globules [231, leading to an increase in its subsequent hydroIysis by pancreatic li-

152 pase [24]. However, preduodenal lipases are subjected to strong inhibition by free fatty acids that can be prevented by the removal of them [23.35]. Extensive studies ‘in vitro’ using bovine serum albumin as fatty acid acceptor have been carried out. However, there is a near complete lack of information on the fatty acid binding ability of other dietary proteins which could be of great importance in the stimulation of the activity of gastric lipolysis. The aim of this work has been to study the effect of ruminant P-lactoglobulin on the lipolytic activity of ruminant pregastric lipase and to elucidate the putative biological role of this protein. In addition, the apparent association constants of p-lactoglobulin for the major fatty acids naturally bound to this protein have also been determined. Materials

and Methods

Isolation of P-lactoglobulin p-lactoglobulin was isolated from bovine milk as described by Perez et al. [9]. Milk was skimmed by centrifugation at 2000 X g at 4 o C for 15 min and whey was obtained by ultrafiltration in an Amicon DC 2A system fitted with a hollow fiber cartridge, Diaflo HlPlOO. This system separates whey proteins with molecular weights below 100 000. The ultrafiltrate was further concentrated in ultrafiltration cells with PM-10 Diaflo Amicon membranes and chromatographed in a Sephadex G-100 (Pharmacia, Uppsala, Sweden) column (90 x 5 cm) equilibrated with 0.025 M sodium acetate, 0.05 M NaCl buffer (pH 6.5) at 4 o C. Fractions enriched in p-lactoglobulin were pooled and rechromatographed in the same conditions. Protein obtained was 97-99% pure by polyacrylamide-agarose gel electrophoresis. No albumin could be detected by immunodiffusion and immunoelectrophoresis.

Determination of the apparent affinity constants Solutions of palmityl and oleyl-carboxy-[ “Clacids (11.2 and 58 mCi/mmol, respectively) (Amersham International, Amersham, U.K.) in heptane were washed three times with 0.01 M potassium phosphate, 0.15 M NaCl buffer (pH 7.2) to eliminate any water soluble contaminants. Fatty acids naturally bound to P-lactoglobulin were eliminated by charcoal treatment [26]. Binding constants and number of binding sites for the interaction between P-lactoglobulin and fatty acids were determined by an equilibrium partition method [27,28]. 600 ~1 of delipidated P-lactoglobulin solution (83 PM) in 0.01 M potassium phosphate, 0.15 M NaCl buffer tpH 7.2) was incubated with 600 ~1 of solution of labelled fatty acid (10-2000 PM) in heptane overnight with agitation at 37 “C. Radioactivity was determined in 200 ~1 samples of organic and aqueous phases by liquid scintillation counting. The partition

ratio of free fatty acids between heptane and protein free buffered salt solution was determined under the same conditions. The binding parameters were calculated using the graphic method of Scatchard by a computerized procedure kindly supplied by Dr. Crisponi, Instituto di Chimica Generale, Cagliari, Italy [29].

Enzymatic assay Tissues from l-2 week old lambs were obtained from the local slaughterhouse. The pharyngeal mucosa lining the opening of the esophagus was dissected and placed on ice. Tissue samples were minced and homogenized in 5 vol. of 0.15 M NaCl. Homogenates were centrifuged for 30 min at 40000 X g and the supernatant was used as crude enzyme preparation. Enzymatic analysis was carried out basically according to Blackberg et al. [30]. 30 mg of tripalmitin (Sigma. London. England) was mixed with 1.3 X 10' cpm of glycerol tri(l-[‘3C]) palmitate (56 ~Ci/mmol) (Amersham International, Amersham, U.K.) and 7.5 ml of arabic gum (2.5%. w/v) in 0.036 M sodium citrate 0.36 M NaCl buffer (pH 5.8) and the mixture was sonicated for 7 min. Then, 150 ~1 of this emulsion and 150 ~1 of bovine serum albumin or p-lactoglobuiin solutions in the same buffer were incubated with 100 ~1 of enzyme extract at 37°C for 1 h. The final concentration of these proteins ranged from 5 to 60 mg/ml. Then, the reaction was stopped by adding 3.25 ml of a mixture containing methanol-chloroformheptane ( 1.4 1/ 1.25,’ 1 v/v) and 0.85 ml of 0.1 M potassium carbonate buffer (pH 10.5) was added. Radioactivity associated with the fatty acids was determined as described in Blackberg et al. [30]. Results

and Discussion

The Scatchard plots obtained for the interaction of bovine p-lactoglobulin with palmitic and olefc acid are shown in Fig. 1. These data show a non linear correlation indicating that P-lactoglobulin contains more than one class of binding sites. In this work, the concentrations of fatty acids and protein corresponded mainly to the range in which the binding sites with high affinity are involved. Thus, the association constants for these sites have been shown. p-lactoglobulin displays one apparent high affinity site for binding of fatty acids whose association constants have been of similar order of magnitude for the two fatty acids studied (4.2 x 10h and 2.3 x lo6 M-’ for palmitic and oleic acids, respectively). The existence of one high affinity site is in accordance with the amount of fatty acids naturally bound to this protein [9]. The values of the constants for interaction of fatty acids with P-lactoglobulin obtained here are higher than those previously reported [S]. As /?-lactoglobulin has fatty acids physiologically

153 bound and the amount present probably depends on the isolation technique, these differences could be explained because they did not use delipidated protein. Thus, fatty acids bound to the protein could have influenced the determination of apparent association constants. The number of high affinity binding sites for fatty acids with p-lactoglobulin and the values of apparent association constants are lower than those of albumin [27,31]. However, the molar concentration of P-lactoglobulin in milk is much higher than that of albumin (about 30 times higher), and therefore P-lactoglobulin is the main fatty acid binding protein in the ruminant whey [2,32]. On the other hand, the effects of p-lactoglobulin and albumin on the pregastric lipase activity are shown in Fig. 2. Optimal assay conditions were obtained using a pharyngeal extract as enzyme source [15]. The amount of fatty acid released was linear with time within an hour of incubation. Initial experiments showed that 87% of the fatty acid released but only 0.3% of the triglycerides, were extracted into the upper phase (data not shown). Taking the lipase activity in protein free buffered salt solutions as 100% (about 22 nmol of fatty acids released per min per ml of pharyngeal extract), addition of p-lactoglobulin or albumin to the incubation mixture increased the lipase activity. Incubation

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Fig. 1. Scatchard plots obtained for the binding of palmitic (A) and oleic (B) acids to bovine @-lactoglobulin. 600 ~1 of delipidated P-lactoglobulin solution (83 PM) in 0.05 M potassium phosphate, 0.15 M NaCl buffer, pH 7.2 as incubated overnight with 600 11 of labelled fatty acid solution (10-2000 PM) in heptane at 37 ’ C, with agitation and the binding measurements were made by the equilibrium partition method. Radioactivity was determined in 200 ~1 samples of organic and aqueous phases by liquid scintillation. v, the concentration of fatty acid bound to the protein per protein concentration; c, the concentration of free fatty acid in the aqueous phase. Each value represents the average of three experiments.

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Protein concentration (mg/ml) Fig. 2. Effect of the bovine p-lactoglobulin and serum albumin on ovine pregastric lipase activity. One hundred ~1 of pregastric lipase extract was incubated with 150 ~1 of glycerol tri(1 -[‘4C]) palmitate (1.3~ lo7 c.p.m. and 4 mg/ml) emulsion in 0.036 M sodium citrate, 0.36 M NaCl buffer, pH 5.8 and 150 ~1 of bovine p-lactoglobulin and serum albumin (S-60 mg/ml final concentration) in the same buffer at 37 “C, for 1 h, at which time, 3.25 ml of a mixture containing methanol-chloroform-heptane (1.41/1.25/1.00 v/v) and 0.85 ml of 0.1 M potassium carbonate buffer, pH 10.5 were added. Radioactivity associated with fatty acids was determined in upper phase by liquid scintillation counting. Enzymatic activity is expressed as the percentage of a control incubated in the absence of added proteins. Each value represents the average for three experiments, each done in duplicate. P-lactoglobulin co), albumin ( A ). Statistical significance of the differences between data was assessed by a paired t-test: /?-lactoglobulin and albumin vs controls P < 0.05 except for albumin at 60 mg/ml; p-lactoglobulin vs albumin P < 0.05 above 10 mg/ml.

with 10 mg/ml, 20 mg/ml and 40 mg/ml of &lactoglobulin gave enzyme activities of approximately 200%, 250% and 190%, respectively. At protein concentration above 20 mg/ml, the increase of lipase activity is lower. This may be explained by assuming that two contrary effects are present: stimulation of lipase activity due to removal of free fatty acids from the site of reaction and inhibition of lipase activity at high concentrations due to the steric effects of protein molecules which block the interface oil-water [25]. Our results show that bovine P-lactoglobulin at the concentration normally present in milk during the first days of lactation [2] markedly increases the activity of pregastric lipase. However, the concentrations of albumin required for a stimulatory effect on lipase activity is greater than that found either in colostrum or in milk of ruminants [32]. Bovine a-lactalbumin, the other major whey protein in ruminant milk, does not have any effect on the pharyngeal lipase activity at concentrations between 5 mg/ml and 40 mg/ml. The efficient digestion and absorption of dietary fat in the newborn is probably the result of the combined action of intragastric and intestinal lipolysis by preduodenal and pancreatic lipases, respectively [24]. Intragastric lipolysis compensates for low pancreatic lipase levels and the products of lipolysis, mainly fatty acids and diacylglycerols, compensate for low bile salts levels, in promoting emulsification of the lipid mixture [24,331. Furthermore, initial digestion of milk fat by

154 preduodenal lipase could markedly enhance its hydrolysis, as intact milk fat globules are not a good substrate for pancreatic lipase [23]. In particular, adult ruminants consume low fat diets and are not particularly well adapted to fat digestion. Lipolytic activity in oral tissues is high in young calves and declines sharply as the animal becomes older. Thus, the higher lipolytic activity in suckling calves is probably a temporary adaptation to the high fat content of milk [16]. Gastric lipolysis is of particular importance in calves because about 47%’ of milk fat which enters the ileum is digested to partial glycerides and fatty acids after diversion of pancreatic secretion, and about 70% of long chain fatty acids are absorbed in the absence of pancreatic juice [34]. Recent observations suggest that free fatty acids may be a lipolysis-promoting factor for some lipases [16,351. Thus, fatty acids liberated by pancreatic colipase-dependent lipase are incorporated into bile acid micelles, facilitating emulsification of water insoluble triglycerides in the intestine [35]. However, preduodenal lipases are inhibited by free fatty acids and their activity is stimulated by addition of albumin, which strongly binds the fatty acids released during lipolysis [23,251. These observations suggest that some dietary proteins could play an important role in gastric lipolysis as fatty acid acceptors. P-lactoglobulin is a milk protein very stable to low pH [36] and resistant to proteolysis by digestive enzymes [37]. These characteristics, coupled with the high concentration of this protein during the colostral period [2], its capacity to bind fatty acids [9] and its ability to increase the activity of pregastric lipase indicate that ruminant p-lactoglobulin could participate in fat digestion during the neonatal period. The existence in milk from other species of proteins able to bind fatty acids, other than P-lactoglobulin and albumin, has not been described. However, high concentrations of milk albumin have been reported in rodent milk, reaching 5 mg/ml in the rat [38] and 20 mg/ml in the mouse [39]. This fact suggests that albumin could participate efficiently in promoting the lingual lipase activity in these species, whose milk is devoid of p-lactoglobulin. Triglycerides of lagomorph milk have a very high content of medium chain fatty acids, particularly octanoic and decanoic acids [40]. Studies have shown that intragastric hydrolysis of milk fat is followed by selective absorption of medium chain fatty acids directly from the gastric mucosa [41] and consequently intragastric lipolysis in these species would not require the presence of the fatty acid binding protein. In human and other primate milk, a potent bile salt-stimulated lipase has been isolated which is stable at pH 3.5 and resists degradation in the stomach [42,43]. This lipase is an important compensatory factor in the intestinal digestion of milk fat in the human

newborn 123,441. This acid binding proteins.

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Acknowledgements We are very grateful to Dr. J. Burgos and Dr. J. Brock for helpful comments. This work was supported by Grants PB87-0642 and ALI 90-0368 from DGICYT. References 1 Godovac-Zimmermann, srnschaft 32. 194-297.

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